Sirtuin1 Maintains Actin Cytoskeleton by Deacetylation of Cortactin in Injured Podocytes.

Recent studies have highlighted the renoprotective effect of sirtuin1 (SIRT1), a deacetylase that contributes to cellular regulation. However, the pathophysiologic role of SIRT1 in podocytes remains unclear. Here, we investigated the function of SIRT1 in podocytes. We first established podocyte-specific Sirt1 knockout (SIRT1(pod-/-)) mice. We then induced glomerular disease by nephrotoxic serum injection. The increase in urinary albumin excretion and BUN and the severity of glomerular injury were all significantly greater in SIRT1(pod-/-) mice than in wild-type mice. Western blot analysis and immunofluorescence showed a significant decrease in podocyte-specific proteins in SIRT1(pod-/-) mice, and electron microscopy showed marked exacerbation of podocyte injury, including actin cytoskeleton derangement in SIRT1(pod-/-) mice compared with wild-type mice. Protamine sulfate-induced podocyte injury was also exacerbated by podocyte-specific SIRT1 deficiency. In vitro, actin cytoskeleton derangement in H2O2-treated podocytes became prominent when the cells were pretreated with SIRT1 inhibitors. Conversely, this H2O2-induced derangement was ameliorated by SIRT1 activation. Furthermore, SIRT1 activation deacetylated the actin-binding and -polymerizing protein cortactin in the nucleus and facilitated deacetylated cortactin localization in the cytoplasm. Cortactin knockdown or inhibition of the nuclear export of cortactin induced actin cytoskeleton derangement and dissociation of cortactin from F-actin, suggesting the necessity of cytoplasmic cortactin for maintenance of the actin cytoskeleton. Taken together, these findings indicate that SIRT1 protects podocytes and prevents glomerular injury by deacetylating cortactin and thereby, maintaining actin cytoskeleton integrity.

Transcriptional landscape of glomerular parietal epithelial cells.

Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire.

Expression of a novel stress-inducible protein, sestrin 2, in rat glomerular parietal epithelial cells.

Sestrin 2, initially identified as a p53 target protein, accumulates in cells exposed to stress and inhibits mammalian target of rapamycin (mTOR) signaling. In normal rat kidneys, sestrin 2 was selectively expressed in parietal epithelial cells (PECs), identified by the marker protein gene product 9.5. In adriamycin nephropathy, sestrin 2 expression decreased in PECs on day 14, together with increased expression of phosphorylated S6 ribosomal protein (P-S6RP), a downstream target of mTOR. Sestrin 2 expression was markedly decreased on day 42, coinciding with glomerulosclerosis and severe periglomerular fibrosis. In puromycin aminonucleoside nephropathy, decreased sestrin 2 expression, increased P-S6RP expression, and periglomerular fibrosis were observed on day 9, when massive proteinuria developed. These changes were transient and nearly normalized by day 28. In crescentic glomerulonephritis, sestrin 2 expression was not detected in cellular crescents, whereas P-S6RP increased. In conditionally immortalized cultured PECs, the forced downregulation of sestrin 2 by short hairpin RNA resulted in increased expression of P-S6RP and increased apoptosis. These data suggest that sestrin 2 is involved in PEC homeostasis by regulating the activity of mTOR. In addition, sestrin 2 could be a novel marker of PECs, and decreased expression of sestrin 2 might be a marker of PEC injury.

Galacto-oligosaccharides attenuate renal injury with microbiota modification.

Tubulointerstitial injury is central to the progression of end-stage renal disease. Recent studies have revealed that one of the most investigated uremic toxins, indoxyl sulfate (IS), caused tubulointerstitial injury through oxidative stress and endoplasmic reticulum (ER) stress. Because indole, the precursor of IS, is synthesized from dietary tryptophan by the gut microbiota, we hypothesized that the intervention targeting the gut microbiota in kidney disease with galacto-oligosaccharides (GOS) would attenuate renal injury. After 2 weeks of GOS administration for 5/6 nephrectomized (Nx) or sham-operated (Sham) rats, cecal indole and serum IS were measured, renal injury was evaluated, and the effects of GOS on the gut microbiota were examined using pyrosequencing methods. Cecal indole and serum IS were significantly decreased and renal injury was improved with decreased infiltrating macrophages in GOS-treated Nx rats. The expression levels of ER stress markers and apoptosis were significantly increased in the Nx rats and decreased with GOS. The microbiota analysis indicated that GOS significantly increased three bacterial families and decreased five families in the Nx rats. In addition, the analysis also revealed that the bacterial family Clostridiaceae was significantly increased in the Nx rats compared with the Sham rats and decreased with GOS. Taken altogether, our data show that GOS decreased cecal indole and serum IS, attenuated renal injury, and modified the gut microbiota in the Nx rats, and that the gut microbiota were altered in kidney disease. GOS could be a novel therapeutic agent to protect against renal injury.

Successful use of intravenous cyclophosphamide pulse therapy for interstitial lung disease in a patient with systemic sclerosis on hemodialysis.

Interstitial lung disease and scleroderma renal crisis are major complications of systemic sclerosis, which occasionally coexist in patients with the diffuse cutaneous subtype. We herein report a case of diffuse cutaneous systemic sclerosis under hemodialysis due to a previous history of scleroderma renal crisis, whose interstitial lung disease was effectively and safely treated with a half dose of i.v. cyclophosphamide pulse. The dose of cyclophosphamide and the timing of hemodialysis leading to efficacy and low toxicity are discussed.

Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.

Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age-related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age-related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid-age (53 weeks old) wild-type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age-related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Age-related impairment of endothelium-dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium-independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid-age WT rats, but not in mid-age GLO1 Tg rats. Age-related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age-related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long-term impact of endothelial dysfunction on vascular aging.

Sperm-associated antigen 4, a novel hypoxia-inducible factor 1 target, regulates cytokinesis, and its expression correlates with the prognosis of renal cell carcinoma.

Hypoxia plays a crucial role in many pathophysiological conditions, including cancer biology, and hypoxia-inducible factor (HIF) regulates transcriptional responses under hypoxia. To elucidate the cellular responses to hypoxia, we performed chromatin immunoprecipitation with deep sequencing in combination with microarray analysis and identified HIF-1 targets. We focused on one of the novel targets, sperm-associated antigen 4 (SPAG4), whose function was unknown. SPAG4, an HIF-1-specific target, is up-regulated in various cultured cells under hypoxia. Examination of SPAG4 expression using a tissue microarray consisting of 190 human renal cell carcinoma (RCC) samples revealed that SPAG4 is an independent prognostic factor of cancer-specific mortality. Live-cell imaging revealed localization of SPAG4 at the intercellular bridge in telophase. We also studied cells in which SPAG4 was knocked down. Hypoxia enhances tetraploidy, which disturbs cell proliferation, and knockdown of SPAG4 increased tetraploid formation and decreased cell proliferation under both normoxic and hypoxic conditions. Studies using deletion mutants of SPAG4 also suggested the involvement of SPAG4 in cytokinesis. Microarray analysis confirmed dysregulation of cytokinesis-related genes by knockdown of SPAG4. In conclusion, SPAG4 is an independent prognostic factor in RCC and plays a crucial role in cytokinesis to defend against hypoxia-induced tetraploid formation. This defensive mechanism may promote survival of cancer cells under hypoxic conditions, thus leading to poor prognosis.

Aberrant Notch1-dependent effects on glomerular parietal epithelial cells promotes collapsing focal segmental glomerulosclerosis with progressive podocyte loss.

Collapsing focal segmental glomerulosclerosis (cFSGS) is a progressive kidney disease characterized by glomerular collapse with epithelial hyperplasia. Here we used a transgenic mouse model of cFSGS with immunotoxin-induced podocyte-specific injury to determine the role for Notch signaling in its pathogenesis. The mice exhibited progressive loss of podocytes and severe proteinuria concomitant with histological features of cFSGS. Hyperplastic epithelium was negative for genetic podocyte tags, but positive for the parietal epithelial cell marker claudin-1, and expressed Notch1, Jagged1, and Hes1 mRNA and protein. Enhanced Notch mRNA expression induced by transforming growth factor-ß1 in cultured parietal epithelial cells was associated with mesenchymal markers (α-smooth muscle actin, vimentin, and Snail1). Notch inhibition in vitro suppressed these phenotypic transcripts and Notch-dependent cell migration. Moreover, Notch inhibition in vivo significantly decreased parietal epithelial cell lesions but worsened proteinuria and histopathology in our cFSGS model. Thus, aberrant Notch1-mediated parietal epithelial cell migration with phenotypic changes appears to underlie the pathogenesis of cFSGS. Parietal epithelial cell hyperplasia may also represent an adaptive response to compensate for a disrupted filtration barrier with progressive podocyte loss.

Albumin-induced apoptosis of glomerular parietal epithelial cells is modulated by extracellular signal-regulated kinase 1/2.

BACKGROUND: The biological role(s) of glomerular parietal epithelial cells (PECs) is not fully understood in health or disease. Given its location, PECs are constantly exposed to low levels of filtered albumin, which is increased in nephrotic states. We tested the hypothesis that PECs internalize albumin and increased uptake results in apoptosis. METHODS: Confocal microscopy of immunofluorescent staining and immunohistochemistry were used to demonstrate albumin internalization in PECs and to quantitate albumin uptake in normal mice and rats as well as experimental models of membranous nephropathy, minimal change disease/focal segmental glomerulosclerosis and protein overload nephropathy. Fluorescence-activated cell sorting analysis was performed on immortalized cultured PECs exposed to fluorescein isothiocyanate (FITC)-labeled albumin in the presence of an endosomal inhibitor or vehicle. Apoptosis was measured by Hoechst staining in cultured PECs exposed to bovine serum albumin. Levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2) were restored by retroviral infection of mitogen-activated protein kinase (MEK) 1/2 and reduced by U0126 in PECs exposed to high albumin levels in culture and apoptosis measured by Hoechst staining. RESULTS: PECs internalized albumin normally, and this was markedly increased in all of the experimental disease models (P<0.05 versus controls). Cultured immortalized PECs also internalize FITC-labeled albumin, which was reduced by endosomal inhibition. A consequence of increased albumin internalization was PEC apoptosis in vitro and in vivo. Candidate signaling pathways underlying these events were examined. Data showed markedly reduced levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in PECs exposed to high albumin levels in nephropathy and in culture. A role for ERK1/2 in limiting albumin-induced apoptosis was shown by restoring p-ERK1/2 by retroviral infection, which reduced apoptosis in cultured PECs, while a forced decrease of p-ERK1/2 through inhibition of MEK 1/2 significantly increased albumin-induced PEC apoptosis. CONCLUSIONS: A normal role of PECs is to take up filtered albumin. However, this is increased in proteinuric glomerular diseases, leading to apoptosis through changes in ERK1/2.

p35, the non-cyclin activator of Cdk5, protects podocytes against apoptosis in vitro and in vivo.

Cyclin-dependent kinase-5 is widely expressed and predominantly regulated by the non-cyclin activator p35. Since we recently showed that expression of p35 in the kidney is restricted to podocytes, we examined here its function in mice in which p35 was genetically deleted. The mice did not exhibit kidney abnormalities during glomerular development or during adult life. Conditionally immortalized cultured podocytes, derived from these null mice, did not have any change in their morphology, differentiation, or proliferation. However, when these cultured podocytes were exposed to UV-C irradiation, serum depletion, puromycin aminonucleoside, or transforming growth factor-beta-1, they showed increased apoptosis compared to those from wild-type mice. Levels of Bcl-2 were decreased in these null podocytes but increased after transduction with human p35. Restoration of p35 or the ectopic expression of Bcl-2 reduced the susceptibility of p35-null podocytes to apoptosis. Experimental glomerulonephritis, characterized by podocyte apoptosis and subsequent crescent formation, was utilized to test these findings in vivo. Podocyte apoptosis was significantly increased in diseased p35-null compared with wild-type mice, accompanied by increased glomerulosclerosis and decreased renal function. Our study shows that p35 does not affect glomerulogenesis but controls podocyte survival following injury, in part, by regulating Bcl-2 expression.

De novo expression of podocyte proteins in parietal epithelial cells during experimental glomerular disease.

Studies have shown that certain cells of the glomerular tuft begin to express proteins considered unique to other cell types upon injury. Little is known about the response of parietal epithelial cells (PEC) to injury. To determine whether PECs change their phenotype upon injury to also express proteins traditionally considered podocyte specific, the following four models of glomerular disease were studied: the transforming growth factor (TGF)-beta1 transgenic mouse model of global glomerulosclerosis, the adriamycin model of focal segmental glomerulosclerosis (FSGS), the anti-glomerular basement membrane (GBM) model of crescentic glomerulonephritis, and the passive Heymann nephritis model of membranous nephropathy. Double immunostaining was performed with antibodies to podocyte-specific proteins (synaptopodin and Wilms' tumor 1) and antibodies to PEC specific proteins (paired box gene 8 and claudin-1). No double staining was detected in normal mice. In contrast, the results showed a statistical increase in the number of cells attached to Bowman basement membrane that were double-positive for both podocyte/PEC proteins in TGF-beta1 transgenic, anti-GBM, and membranous animals. Double-positive cells for both podocyte and PEC proteins were also statistically increased in the glomerular tuft in TGF-beta1 transgenic, anti-GBM, and FSGS mice. These results are consistent with glomerular cells coexpressing podocyte and PEC proteins in experimental glomerular disease, but not under normal circumstances.

The enigmatic parietal epithelial cell is finally getting noticed: a review.

Although the normal glomerulus comprises four resident cell types, least is known about the parietal epithelial cells (PECs). This comprehensive review addresses the cellular origin of PECs, discusses the normal structure and protein makeup of PECs, describes PEC function, and defines the responses to injury in disease and how these events lead to clinical events. The data show that PECs have unique properties and that new functions are being recognized such as their role in differentiating into podocytes during disease.

A new function for parietal epithelial cells: a second glomerular barrier.

The functional role of glomerular parietal epithelial cells (PECs) remains poorly understood. To test the hypothesis that PECs form an impermeable barrier to filtered protein through the formation of tight junctions (TJ), studies were performed in normal animals and in the anti-glomerular basement membrane (GBM) model of crescentic nephritis. Electron microscopy showed well-defined TJ between PECs in normal mice, which no longer could be identified when these cells became extensively damaged or detached from their underlying Bowman's basement membrane. The TJ proteins claudin-1, zonula occludens-1, and occludin stained positive in PECs; however, staining decreased in anti-GBM disease. To show that these events were associated with increased permeability across the PEC-Bowman's basement membrane barrier, control and diseased animals were injected intravenously with either Texas red-conjugated dextran (3 kDa) or ovalbumin (45 kDa) tracers. As expected, both tracers were readily filtered across the glomerular filtration barrier and taken up by proximal tubular cells. However, when the glomerular filtration barrier was injured in anti-GBM disease, tracers were taken up by podocytes and PECs. Moreover, tracers were also detected between PECs and the underlying Bowman's basement membrane, and in many instances were detected in the extraglomerular space. We propose that together with its underlying Bowman's basement membrane, the TJ of PECs serve as a second barrier to protein. When disturbed following PEC injury, the increase in permeability of this layer to filtered protein is a mechanism underlying periglomerular inflammation characteristic of anti-GBM disease.

Inducible rodent models of acquired podocyte diseases.

Glomerular diseases remain the leading cause of chronic and end-stage kidney disease. Significant advances in our understanding of human glomerular diseases have been enabled by the development and better characterization of animal models. Diseases of the glomerular epithelial cells (podocytes) account for the majority of proteinuric diseases. Rodents have been extensively used experimentally to better define mechanisms of disease induction and progression, as well as to identify potential targets and therapies. The development of podocyte-specific genetically modified mice has energized the research field to better understand which animal models are appropriate to study acquired podocyte diseases. In this review we discuss inducible experimental models of acquired nondiabetic podocyte diseases in rodents, namely, passive Heymann nephritis, puromycin aminonucleoside nephrosis, adriamycin nephrosis, liopolysaccharide, crescentic glomerulonephritis, and protein overload nephropathy models. Details are given on the model backgrounds, how to induce each model, the interpretations of the data, and the benefits and shortcomings of each. Genetic rodent models of podocyte injury are excluded.

Establishment of conditionally immortalized mouse glomerular parietal epithelial cells in culture.

Parietal epithelial cells (PEC) are major constituents of crescents in crescentic glomerulonephritis. The purpose of these studies was to establish an immortalized PEC cell line with similar characteristics to PEC in vivo for use in future mechanistic studies. Glomeruli were isolated from H-2Kb tsA58 transgenic mice (ImmortoMouse) by standard differential sieving, and several candidate PEC cell lines were obtained by subcloning outgrowths of cells from capsulated glomeruli. One clone, designated mouse PEC (mPEC), was extensively characterized. mPEC exhibited a compact cell body with typical epithelial morphology when grown in permissive conditions, but the cell shape changed to polygonal after 14 d in growth-restrictive conditions. mPEC but not podocytes used as a negative control expressed claudin-1, claudin-2, and protein gene product 9.5, which are proteins specific to PEC in vivo, and did not express the podocyte-specific proteins synaptopodin and nephrin. The junctional proteins zonula occludens-1 and beta-catenin stained positively in both mPEC and podocytes, but the staining pattern at cell-cell contacts was intermittent in mPEC and linear in podocytes. Finally, mPEC had thin bundled cortical F-actin filaments and no F-actin projections compared with podocytes, which exhibited thick bundled cortical F-actin filaments and interdigitating F-actin projections at cell-cell contacts. We conclude that immortalized mPEC in culture exhibit specific features of PEC in vivo and that these cells are distinct from podocytes, despite having the same mesenchymal origin. This mPEC line will assist in future mechanistic studies of PEC and enhance our understanding of glomerular injury.

Induction of TRPC6 channel in acquired forms of proteinuric kidney disease.

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene that codes for a slit diaphragm-associated, cation-permeable ion channel have been shown recently to co-segregate with hereditary forms of progressive kidney failure. Herein is shown that induced expression of wild-type TRPC6 is a common feature of human proteinuric kidney diseases, with highest induction observed in membranous nephropathy. Cultured podocytes that are exposed to complement upregulate TRPC6 protein. Stimulation of receptor-operated channels in puromycin aminonucleoside-treated podocytes leads to increased calcium influx in a time- and dosage-dependent manner. Mechanistically, it is shown that TRPC6 is functionally connected to the podocyte actin cytoskeleton, which is rearranged upon overexpression of TRPC6. Transient in vivo gene delivery of TRPC6 into mice leads to expression of TRPC6 protein at the slit diaphragm and causes proteinuria. These studies suggest the involvement of TRPC6 in the pathology of nongenetic forms of proteinuric disease.

Hypoxia and expression of hypoxia-inducible factor in the aging kidney.

Renal senescence is characterized by interstitial fibrosis and loss of peritubular capillaries. In this study, we provided evidence of tubulointerstitial hypoxia and the operation of hypoxia-inducible factor (HIF) in the aging kidney. Using two distinct methods, pimonidazole immunostaining and the expression of the "hypoxia-responsive" reporter of the transgenic rats, we identified the age-related expansion of hypoxia in all areas of the kidney. Expansion was most prominent in the cortex. Clusters of hypoxic tubules were observed in the superficial cortical zones, areas adjacent to the outer nephrons and expanded in the medullary rays. The degree of hypoxia was positively correlated with the age-related tubulointerstitial injury (R(2) = 0.88, p <.01), which was associated with the upregulation of HIF-regulated genes, such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT1) (real-time polymerase chain reaction). These findings point to the involvement of hypoxia and highlight the pathological relevance of HIF and its target genes in the aging kidney.

Novel drugs and the response to hypoxia: HIF stabilizers and prolyl hydroxylase.

Tissue hypoxia occurs when local metabolism is disturbed by an imbalance between oxygen supply and consumption. This condition can lead to a variety of serious ischemic disorders, including a number of important cardiovascular diseases. In the search for therapeutic approaches, focused modalities which specifically target hypoxia have been particularly sought. These efforts would profit from the ability to utilize the mechanisms by which cells adjust to hypoxic conditions. At the center of the cellular response to hypoxia is hypoxia-inducible factor, HIF. This factor is composed of two subunits, an oxygen-sensitive HIF-alpha subunit and a constitutively expressed HIF-beta subunit. Intracellular accumulation of HIF induces the coordinated expression of a number of adaptive genes against hypoxic insult. Because activation of HIF is a promising therapeutic modality for ischemic cardiovascular disease, recent studies have focused on the development of HIF stimulators. HIF levels are regulated by prolyl hydroxylation and asparaginyl hydroxylation of the HIF-alpha subunit. To date, a single HIF asparaginyl hydroxylase has been identified, factor inhibiting HIF (FIH), whereas the mammalian genome encodes three closely related proteins that have HIF prolyl hydroxylase activity, PHD1, PHD2 and PHD3. Recent patents have disclosed methods for identifying modulators of HIF or PHD as well as novel compounds with properties of HIF modulation or prolyl hydroxylase inhibition. This review highlights the identification of novel HIF stabilizers as specific molecularly targeted therapies against cardiovascular disease.

Induction of protective genes by cobalt ameliorates tubulointerstitial injury in the progressive Thy1 nephritis.

BACKGROUND: We previously demonstrated that chronic hypoxia has pivotal roles in the progression of tubulointerstitial injury from the early stage of the uninephrectomized Thy1 nephritis model. We have also shown that pretreatment of cobalt confers renoprotection in the ischemia/reperfusion (I/R) injury, in association with the up-regulation of hypoxia-inducible factor (HIF)-regulated genes. Here, we tested the hypothesis that cobalt administration not only attenuates acute ischemic insult, but also ameliorates tubulointerstitial injury secondary to chronic hypoxia. METHODS: We applied sustained cobalt treatment to the uninephrectomized Thy1 nephritis model at 3 to 5 weeks, when tubular hypoxia appeared. Histologic evaluation, including glomerular and peritubular capillary networks, was made at 8 weeks. HIF activation was confirmed by real-time polymerase chain reaction (PCR) analyses for HIF-regulated genes, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), and heme oxygenase 1 (HO-1). Up-regulation of HIF-1alpha and HIF-regulated genes was also verified by Western blotting analysis. To elucidate responsible mechanisms of cobalt in the amelioration of tubuloniterstitial injury, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was conducted at 5 weeks. A combination therapy with angiotensin receptor blocker (ARB), olmesartan, was also challenged. RESULTS: Although the intervention did not change glomerular structural damage or urinary protein excretion rate, tubulointerstitial injury was improved in cobalt-treated animals when compared with the vehicle-treated group. The amelioration was associated with the parallel up-regulation of renoprotective, HIF-regulated gene expression. TUNEL staining revealed that the number of apoptotic cells was reduced in the cortex by cobalt administration, suggesting that renoprotection was achieved partly through its antiapoptotic properties. Furthermore, it was demonstrated that cobalt treatment exerts additional renoprotective effects with the ARB treatment in this model. CONCLUSION: Maneuvers to activate HIF in the ischemic tubulointerstitium will be a new direction to future therapeutic strategies.