Chuan-Ming Hao

Thiazolidinediones expand body fluid volume through PPARγ stimulation of ENaC-mediated renal salt absorption

Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-γ (PPARγ, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct–specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg flox/flox mice. Deletion of collecting duct Pparg decreased renal Na+ avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na+ absorption and Scnn1g mRNA (encoding the epithelial Na+ channel ENaCγ) expression through a PPARγ-dependent pathway. These studies identify Scnn1g as a PPARγ target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.

Opposite effects of cyclooxygenase-1 and -2 activity on the pressor response to angiotensin II

Therapeutic use of cyclooxygenase-inhibiting (COX-inhibiting) nonsteroidal antiinflammatory drugs (NSAIDs) is often complicated by renal side effects including hypertension and edema. The present studies were undertaken to elucidate the roles of COX1 and COX2 in regulating blood pressure and renal function. COX2 inhibitors or gene knockout dramatically augment the pressor effect of angiotensin II (Ang II). Unexpectedly, after a brief increase, the pressor effect of Ang II was abolished by COX1 deficiency (either inhibitor or knockout). Ang II infusion also reduced medullary blood flow in COX2-deficient but not in control or COX1-deficient animals, suggesting synthesis of COX2-dependent vasodilators in the renal medulla. Consistent with this, Ang II failed to stimulate renal medullary prostaglandin E(2) and prostaglandin I(2) production in COX2-deficient animals. Ang II infusion normally promotes natriuresis and diuresis, but COX2 deficiency blocked this effect. Thus, COX1 and COX2 exert opposite effects on systemic blood pressure and renal function. COX2 inhibitors reduce renal medullary blood flow, decrease urine flow, and enhance the pressor effect of Ang II. In contrast, the pressor effect of Ang II is blunted by COX1 inhibition. These results suggest that, rather than having similar cardiovascular effects, the activities of COX1 and COX2 are functionally antagonistic.

Sirt1 activation protects the mouse renal medulla from oxidative injury

Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase that exerts many of the pleiotropic effects of oxidative metabolism. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress. Here, we set out to investigate the role of Sirt1 in the kidney. Our initial analysis indicated that it was abundantly expressed in mouse renal medullary interstitial cells in vivo. Knocking down Sirt1 expression in primary mouse renal medullary interstitial cells substantially reduced cellular resistance to oxidative stress, while pharmacologic Sirt1 activation using either resveratrol or SRT2183 improved cell survival in response to oxidative stress. The unilateral ureteral obstruction (UUO) model of kidney injury induced markedly more renal apoptosis and fibrosis in Sirt1+/- mice than in wild-type controls, while pharmacologic Sirt1 activation substantially attenuated apoptosis and fibrosis in wild-type mice. Moreover, Sirt1 deficiency attenuated oxidative stress-induced COX2 expression in cultured mouse renal medullary interstitial cells, and Sirt1+/- mice displayed reduced UUO-induced COX2 expression in vivo. Conversely, Sirt1 activation increased renal medullary interstitial cell COX2 expression both in vitro and in vivo. Furthermore, exogenous PGE2 markedly reduced apoptosis in Sirt1-deficient renal medullary interstitial cells following oxidative stress. Taken together, these results identify Sirt1 as an important protective factor for mouse renal medullary interstitial cells following oxidative stress and suggest that the protective function of Sirt1 is partly attributable to its regulation of COX2 induction. We therefore suggest that Sirt1 provides a potential therapeutic target to minimize renal medullary cell damage following oxidative stress.

Dehydration activates an NF-κB–driven, COX2-dependent survival mechanism in renal medullary interstitial cells

Renal prostaglandin (PG) synthesis is mediated by cyclooxygenase-1 and -2 (COX1 and COX2). After dehydration, the maintenance of normal renal function becomes particularly dependent upon PG synthesis. The present studies were designed to examine the potential link between medullary COX1 and COX2 expression in hypertonic stress. In response to water deprivation, COX2, but not COX1, mRNA levels increase significantly in the renal medulla, specifically in renal medullary interstitial cells (RMICs). Water deprivation also increases renal NF-kappaB-driven reporter expression in transgenic mice. NF-kappaB activity and COX2 expression could be induced in cultured RMICs with hypertonic sodium chloride and mannitol, but not urea. RMIC COX2 expression was also induced by driving NF-kappaB activation with a constitutively active IkappaB kinase alpha (IKKalpha). Conversely, introduction of a dominant-negative IkappaB mutant reduced COX2 expression after hypertonicity or IKKalpha induction. RMICs failed to survive hypertonicity when COX2 was downregulated using a COX2-selective antisense or blocked with the selective nonsteroidal anti-inflammatory drug (NSAID) SC58236, reagents that did not affect cell survival in isotonic media. In rabbits treated with SC58236, water deprivation induced apoptosis of medullary interstitial cells in the renal papilla. These results demonstrate that water deprivation and hypertonicity activate NF-kappaB. The consequent increase in COX2 expression favors RMIC survival in hypertonic conditions. Inhibition of RMIC COX2 could contribute to NSAID-induced papillary injury.

Prostaglandin E2-EP4 Receptor Promotes Endothelial Cell Migration via ERK Activation and Angiogenesis in Vivo

Prostaglandin E2 (PGE2), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1–4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4flox/flox mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE2 or the EP4 selective agonists PGE1-OH and ONO-AE1–329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4flox/flox cells, no effects were seen in adenoCre-transduced EP4flox/flox cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE2-induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE2, as well as PGE1-OH and ONO-AE1–329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.

Differential Expression of the Intermediate Filament Protein Nestin during Renal Development and Its Localization in Adult Podocytes

Nestin, an intermediate filament protein, is widely used as stem cell marker. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. These studies examined renal nestin localization and developmental expression in mice. In developing kidney, anti-nestin antibody revealed strong immunoreactivity in vascular cleft of the S-shaped body and vascular tuft of capillary loop-stage glomerulus. The nestin-positive structures also were labeled by endothelial cell markers FLK1 and CD31 in immature glomeruli. Nestin was not detected in epithelial cells of immature glomeruli. In contrast, in mature glomerular, nestin immunoreactivity was observed only outside laminin-positive glomerular basement membrane, and co-localized with nephrin, consistent with podocyte nestin expression. In adult kidney, podocytes were the only cells that exhibited persistent nestin expression. Nestin was not detected in ureteric bud and its derivatives throughout renal development. Cell lineage studies, using a nestin promoter-driven Cre mouse and a ROSA26 reporter mouse, showed a strong beta-galactosidase activity in intermediate mesoderm in an embryonic day 10 embryo and all of the structures except those that were derived from ureteric bud in embryonic kidney through adult kidney. These studies show that nestin is expressed in progenitors of glomerular endothelial cells and renal progenitors that are derived from metanephric mesenchyme. In the adult kidney, nestin expression is restricted to differentiated podocytes, suggesting that nestin could play an important role in maintaining the structural integrity of the podocytes.

Selective targeting of cyclooxygenase-2 reveals its role in renal medullary interstitial cell survival

Renal medullary interstitial cells (MICs) are a major site of cyclooxygenase (COX)-mediated PG synthesis. These studies examined the role of COX in MIC survival. Immunoblot and nuclease protection demonstrate that cultured MICs constitutively express COX2, with little constitutive COX1 expression. SC-58236, a COX2-selective inhibitor, but not SC-58560, a COX1 inhibitor, preferentially blocks PGE2 synthesis in MICs. Transduction with a COX2 antisense adenovirus reduced MIC COX2 protein expression and also decreased PGE2production. Antisense downregulation of COX2 was associated with MIC death, whereas a control adenovirus was without effect. Similarly, the COX2-selective inhibitor SC-58236 (30 μM) and several nonselective COX-inhibiting nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac, ibuprofen, and indomethacin, all caused MIC death. In contrast, SC-58560, a COX1-selective inhibitor, was 100-fold less potent for inducing MIC death than its structural congener SC-58236. NSAID-induced MIC death was associated with DNA laddering and nuclear fragmentation, consistent with apoptosis. These results suggest that COX2 plays an important role in MIC survival. COX2 inhibition may contribute to NSAID-associated injury of the renal medulla.

Overexpression of Cyclooxygenase-2 Predisposes to Podocyte Injury

Increased podocyte cyclooxygenase-2 (COX-2) expression is seen in rats after renal ablation and Thy-1 nephritis and in cultured murine podocytes in response to mechanical stress. For investigation of whether COX-2 overexpression plays a role in podocyte injury, transgenic B6/D2 mice in which COX-2 expression was driven by a nephrin promoter were established. Selective upregulation of COX-2 expression in podocytes of transgenic mouse kidneys was confirmed by immunoblotting and immunohistochemistry. Whether upregulation of podocyte-specific COX-2 expression enhanced sensitivity to the development of Adriamycin nephropathy was examined. Adriamycin administration induced dramatically more albuminuria and foot process effacement and reduced glomerular nephrin mRNA and immunoreactivity in transgenic mice compared with wild-type littermates. Adriamycin also markedly increased immunoreactive COX-2 expression in podocytes from transgenic mice compared with the wild-type mice. Reverse transcriptase-PCR indicated that this increase represented a stimulation of endogenous COX-2 mRNA expression rather than COX-2 mRNA driven by the nephrin promoter. Balb/C mice, which are susceptible to renal injury by Adriamycin, also increased podocyte COX-2 expression and reduced nephrin expression in response to administration of the drug. Long-term treatment with the COX-2-specific inhibitor SC58236 ameliorated the albuminuria that was induced by Adriamycin in the transgenic mice. SC58236 also reduced Adriamycin-induced foot process effacement in both the COX-2 transgenic mice and Balb/C mice. Therefore, overexpression of COX-2 may predispose podocytes to further injury.

Sirtuins and Their Relevance to the Kidney

Sirtuins (silent information regulator 2 [Sir2] proteins) belong to an ancient family of evolutionary conserved nicotinamide adenine dinucleotide (NAD)(+)-dependent enzymes with deacetylase and/or mono-ADP-ribosyltransferase activity. They regulate DNA repair and recombination, chromosomal stability, and gene transcription, and most importantly mediate the health-promoting effects of caloric restriction (CR), which includes the retardation of aging. At least seven Sir2 homologs, sirtuins (SIRT) 1 to 7 have been identified in mammals. Mammalian SIRT1, the most extensively studied family member, couples protein deacetylation with NAD(+) hydrolysis and links cellular energy and redox state to multiple signaling and survival pathways. Cell-type and context-specific activation of sirtuins increases resistance to metabolic, oxidative, and hypoxic stress in different tissues. In particular, SIRT1 plays a central role in mediating the beneficial effects of CR, and its activation associates with longevity and the attenuation of metabolic disorders. SIRT1 in the kidney is cytoprotective and participates in the regulation of BP and sodium balance. Here, we review sirtuin biology and discuss how CR-triggered sirtuin-dependent pathways affect renal physiology and the pathogenesis of kidney diseases and related disorders.

The histone deacetylase, SIRT1, contributes to the resistance of young mice to ischemia/reperfusion-induced acute kidney injury.

Acute kidney injury (AKI) is a critical condition with a mortality rate as high as 50% and significantly contributes to the burden of end-stage renal disease (ESRD) requiring renal replacement therapy. The incidence and prognosis of AKI have been shown to vary with patient age, with younger individuals being more resistant to AKI. In mice, clamping the renal artery for 45 min causes substantial kidney damage in 4-month-old animals but only mild renal injury in 2-month-old animals. Here, younger mice were found to express higher levels of the NAD(+)-dependent histone deacetylase SIRT1 in the kidney. A small molecule SIRT1 activator, SRT-1720, markedly improved renal tubular pathology and overall renal function in adult mice following ischemia/reperfusion. Genetic ablation of one allele (SIRT1(+/-)) significantly enhanced the level of kidney damage relative to that in wild-type (SIRT1(+/+)) mice. The mechanisms underlying the protective effect of SIRT1 included the suppression of cell apoptosis. Hence, our results suggest that SIRT1 might be a novel therapeutic target for ischemia/reperfusion-induced kidney damage.