Ningjun Li

Production and Actions of Hydrogen Sulfide, a Novel Gaseous Bioactive Substance, in the Kidneys

Hydrogen sulfide (H2S), a novel endogenous gaseous bioactive substance, has recently been implicated in the regulation of cardiovascular and neuronal functions. However, its role in the control of renal function is unknown. In the present study, incubation of renal tissue homogenates with l-cysteine (l-Cys) (as a substrate) produced H2S in a concentration-dependent manner. This H2S production was completely abolished by inhibition of both cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CGL), two major enzymes for the production of H2S, using amino-oxyacetic acid (AOAA), an inhibitor of CBS, and propargylglycine (PPG), an inhibitor of CGL. However, inhibition of CBS or CGL alone induced a small decrease in H2S production. In anesthetized Sprague-Dawley rats, intrarenal arterial infusion of an H2S donor (NaHS) increased renal blood flow, glomerular filtration rate (GFR), urinary sodium (UNa·V), and potassium (UK·V) excretion. Consistently, infusion of both AOAA and PPG to inhibit the endogenous H2S production decreased GFR, UNa·V, and UK·V, and either one of these inhibitors alone had no significant effect on renal functions. Infusion of l-Cys into renal artery to increase the endogenous H2S production also increased GFR, UNa·V, and UK·V, which was blocked by AOAA plus PPG. It was shown that H2S had both vascular and tubular effects and that the tubular effect of H2S might be through inhibition of Na+/K+/2Cl- cotransporter and Na+/K+/ATPase activity. These results suggest that H2S participates in the control of renal function and increases urinary sodium excretion via both vascular and tubular actions in the kidney.

Histone deacetylase 4 selectively contributes to podocyte injury in diabetic nephropathy

Studies have highlighted the importance of histone deacetylase (HDAC)-mediated epigenetic processes in the development of diabetic complications. Inhibitors of HDAC are a novel class of therapeutic agents in diabetic nephropathy, but currently available inhibitors are mostly nonselective inhibit multiple HDACs, and different HDACs serve very distinct functions. Therefore, it is essential to determine the role of individual HDACs in diabetic nephropathy and develop HDAC inhibitors with improved specificity. First, we identified the expression patterns of HDACs and found that, among zinc-dependent HDACs, HDAC2/4/5 were upregulated in the kidney from streptozotocin-induced diabetic rats, diabetic db/db mice, and in kidney biopsies from diabetic patients. Podocytes treated with high glucose, advanced glycation end products, or transforming growth factor-β (common detrimental factors in diabetic nephropathy) selectively increased HDAC4 expression. The role of HDAC4 was evaluated by in vivo gene silencing by intrarenal lentiviral gene delivery and found to reduce renal injury in diabetic rats. Podocyte injury was associated with suppressing autophagy and exacerbating inflammation by HDAC4-STAT1 signaling in vitro. Thus, HDAC4 contributes to podocyte injury and is one of critical components of a signal transduction pathway that links renal injury to autophagy in diabetic nephropathy.

Contribution of guanine nucleotide exchange factor Vav2 to hyperhomocysteinemic glomerulosclerosis in rats.

We currently reported that Vav2, a member of the guanine nucleotide exchange factor-Vav subfamily, participates in homocysteine-induced increases in Rac1 activity and consequent activation of NADPH oxidase in rat mesangial cells. However, the physiological relevance of this cellular action of Vav2 remains unknown. The present study tested a hypothesis that Vav2 importantly mediates the injurious action of homocysteine on glomeruli and thereby contributes to the development of glomerulosclerosis during hyperhomocysteinemia. We found that, among Vav members, Vav2 was abundantly expressed in glomeruli. When Vav2 short hairpin RNA was transfected into the kidneys of Sprague-Dawley rats, hyperhomocysteinemia induced by folate-free diet failed to significantly enhance Rac1 activity and increase NADPH-dependent superoxide production. In these rats with silenced renal Vav2 gene, glomerular injury during hyperhomocysteinemia was markedly attenuated compared with those rats only receiving mock vector transfection, as shown by ameliorated albuminuria and extracellular matrix metabolism. In the rat kidneys with transfection of a dominant-active Vav2 variant (onco-Vav2), we found that overexpression of Vav2 led to significant increases in Rac1 activity, superoxide production, and glomerular injury, which was similar to that induced by hyperhomocysteinemia. However, this Vav2 overexpression was unable to further enhance homocysteine-induced glomerular injury. We concluded that Vav2-mediated activation of NADPH oxidase is an important initiating mechanism resulting in hyperhomocysteinemic glomerular injury through enhanced local oxidative stress.

Hypoxia-inducible factor-1α contributes to the profibrotic action of angiotensin II in renal medullary interstitial cells

To examine whether hypoxia-inducible factor (HIF)-1α mediates the profibrotic effects of angiotensin II, we treated cultured renal medullary interstitial cells with angiotensin II and found that it increased HIF-1α levels. This was accompanied by a significant upregulation of collagen I/III, the tissue inhibitor of metalloproteinase-1, elevation of the proliferation marker proliferating cell nuclear antigen, and a transdifferentiation marker vimentin. All these effects of angiotensin II were completely blocked by siRNA for HIF-1α but not HIF-2α. Overexpression of a prolyl-hydroxylase domain-containing protein 2 (PHD2) transgene, the predominant renal HIF prolyl-hydroxylase, attenuated the effects of angiotensin II and its gene silencing enhanced the effects of angiotensin II. Removal of hydrogen peroxide eliminated angiotensin II-induced profibrotic effects. A 2-week infusion of rats with angiotensin II increased the expression of HIF-1α and α-smooth muscle actin, another marker of transdifferentiation, in renal medullary interstitial cells in vivo. Thus, our study suggests that HIF-1α mediates angiotensin II-induced profibrotic effects through activation of cell transdifferentiation. We propose that redox regulation of prolyl-PHD2 plays a critical role in angiotensin II-induced activation of HIF-1α in renal cells.

Role of Renal Medullary Heme Oxygenase in the Regulation of Pressure Natriuresis and Arterial Blood Pressure

Recent studies have demonstrated that inhibition of renal medullary heme oxygenase (HO) activity and carbon monoxide (CO) significantly decreases renal medullary blood flow and sodium excretion. Given the crucial role of renal medullary blood flow in the control of pressure natriuresis, the present study was designed to determine whether renal medullary HO activity and resulting CO production participate in the regulation of pressure natriuresis and thereby the long-term control of arterial blood pressure. In anesthetized Sprague-Dawley rats, increases in renal perfusion pressure induced significant elevations of CO concentrations in the renal medulla. Renal medullary infusion of chromium mesoporphyrin (CrMP), an inhibitor of HO activity, remarkably inhibited HO activity and the renal perfusion pressure-dependent increases in CO levels in the renal medulla and significantly blunted pressure natriuresis. In conscious Sprague-Dawley rats, continuous infusion of CrMP into the renal medulla significantly increased mean arterial pressure (129±2.5 mm Hg in CrMP group versus 118±1.6 mm Hg in vehicle group) when animals were fed a normal salt diet (1% NaCl). After rats were switched to a high-salt diet (8% NaCl) for 10 days, CrMP-treated animals exhibited further increases in mean arterial pressure compared with CrMP-treated animals that were kept on normal salt diet (152±4.1 versus 130±4.2 mm Hg). These results suggest that renal medullary HO activity plays a crucial role in the control of pressure natriuresis and arterial blood pressure and that impairment of this HO/CO-mediated antihypertensive mechanism in the renal medulla may result in the development of hypertension.

Salt-Sensitive Hypertension Induced by Decoy of Transcription Factor Hypoxia-Inducible Factor-1α in the Renal Medulla

Hypoxia inducible factor (HIF)-1&agr;, a transcription factor, is abundantly expressed in the renal medulla and regulates many oxygen-sensitive genes such as nitric oxide synthase, cyclooxygenase-2, and heme oxygenase-1. Given the important roles of these genes in the control of arterial pressure, the present study was to test the hypothesis that HIF-1&agr;–mediated gene activation serves as an antihypertensive pathway by regulating renal medullary function and sodium excretion. HIF-1&agr; decoy oligodeoxynucleotides (ODNs) or scrambled ODNs were transfected into the renal medulla in uninephrectomized Sprague–Dawley rats. Two weeks after ODN transfection, the HIF-1&agr; binding activities were significantly inhibited by 45%, and high salt–induced increases of nitric oxide synthase-2 and heme oxygenase-1 transcriptions were also inhibited by 70% and 61% in the renal medulla from decoy rats. The natriuretic responses and increases of renal medullary blood flow responding to the elevations of renal perfusion pressure were significantly blunted by 50% and 37% in decoy rats. Intravenously acute sodium loading increased medullary blood flow and urinary sodium excretion, which was remarkably attenuated in decoy rats. In decoy rats, high salt intake caused a greater positive sodium balance. Consequently, arterial pressure was remarkably increased (from 118±1.9 to 154±6.3 mm Hg) in decoy rats but not in control rats when the rats were challenged with a high salt diet. There was no blood pressure change in decoy rats that were maintained in normal salt diet. In conclusion, HIF-1&agr;–mediated gene activation importantly participates in the regulation of renal medullary function and long-term arterial blood pressure.

Silencing of Hypoxia-Inducible Factor-1α Gene Attenuated Angiotensin II–Induced Renal Injury in Sprague-Dawley Rats

Although it has been shown that upregulation of hypoxia-inducible factor (HIF)-1&agr; is protective in acute ischemic renal injury, long-term overactivation of HIF-1&agr; is implicated to be injurious in chronic kidney diseases. Angiotensin II (Ang II) is a well-known pathogenic factor producing chronic renal injury and has also been shown to increase HIF-1&agr;. However, the contribution of HIF-1&agr; to Ang II–induced renal injury has not been evidenced. The present study tested the hypothesis that HIF-1&agr; mediates Ang II–induced renal injury in Sprague-Dawley rats. Chronic renal injury was induced by Ang II infusion (200 ng/kg per minute) for 2 weeks in uninephrectomized rats. Transfection of vectors expressing HIF-1&agr; small hairpin RNA into the kidneys knocked down HIF-1&agr; gene expression by 70%, blocked Ang II–induced HIF-1&agr; activation, and significantly attenuated Ang II–induced albuminuria, which was accompanied by inhibition of Ang II–induced vascular endothelial growth factor, a known glomerular permeability factor, in glomeruli. HIF-1&agr; small hairpin RNA also significantly improved the glomerular morphological damage induced by Ang II. Furthermore, HIF-1&agr; small hairpin RNA blocked Ang II–induced upregulation of collagen and &agr;-smooth muscle actin in tubulointerstitial region. There was no difference in creatinine clearance and Ang II–induced increase in blood pressure. HIF-1&agr; small hairpin RNA had no effect on Ang II–induced reduction in renal blood flow and hypoxia in the kidneys. These data suggested that overactivation of HIF-1&agr;–mediated gene regulation in the kidney is a pathogenic pathway mediating Ang II–induced chronic renal injuries, and normalization of overactivated HIF-1&agr; may be used as a treatment strategy for chronic kidney damages associated with excessive Ang II.

Hypoxia-inducible factor prolyl-hydroxylase-2 mediates transforming growth factor beta 1-induced epithelial-mesenchymal transition in renal tubular cells

Transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGF-β1-induced signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGF-β1-induced EMT using cultured renal tubular cells. Our results showed that TGF-β1 stimulated EMT in renal tubular cells as indicated by the significant decrease in epithelial marker P-cadherin, and the increase in mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1). Meanwhile, we found that TGF-β1 time-dependently increased HIF-1α and that HIF-1α siRNA significantly inhibited TGF-β1-induced EMT, suggesting that HIF-1α mediated TGF-β1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1:PHD2=0.21:1.0). Importantly, PHD2 mRNA and protein, but not PHD1, were decreased by TGF-β1. Furthermore, over-expression of PHD2 transgene almost fully prevented TGF-β1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGF-β1-induced EMT. Finally, Smad2/3 inhibitor SB431542 prevented TGF-β1-induced PHD2 decrease, suggesting that Smad2/3 may mediate TGF-β1-induced EMT through PHD2/HIF-1α pathway. It is concluded that TGF-β1 decreased PHD2 expression via an Smad-dependent signaling pathway, thereby leading to HIF-1α accumulation and then EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGF-β1, and may be a new therapeutic target in chronic kidney diseases.

Podocyte Injury and Glomerulosclerosis in Hyperhomocysteinemic Rats

Background/Aims: We previously reported that increase in plasma homocysteine (Hcys) levels by a 6-week methionine treatment produced remarkable glomerular injury. However, the mechanism by which hyperhomocysteinemia (hHcys) produces glomerular injury remains unknown. The present study was to observe when glomerular injury happens during hHcys and to explore the possible role of podocyte injury in the progression of glomerulosclerosis associated with hHcys. Methods: Uninephrectomized Sprague-Dawley rats treated with methionine were used to examine the time course of glomerular injury induced by hHcys. Results: Creatinine clearance was not different until rats were treated with methionine for 6 weeks, although plasma Hcys levels significantly increased at the 1st week of methionine treatment. However, urinary albumin excretion increased at the 2nd week of methionine treatment. Morphological examinations showed that mesangial expansion occurred at the 2nd week and podocyte effacement was also observed as processed glomerular damage during hHcys. Immunofluorescence analyses demonstrated that podocin and nephrin expressions were reduced, while α-actinin-4 increased during hHcys. Conclusions: Increased plasma Hcys level is an important pathogenic factor resulting in glomerular injury even in the very early time of hHcys. These pathogenic effects of Hcys are associated with podocyte injury and changed expression and distribution of podocyte-associated proteins.

Progranulin protects against renal ischemia/reperfusion injury in mice

Progranulin (PGRN), an autocrine growth factor, has multiple physiological functions and is widely involved in the pathogenesis of many types of diseases. The pivotal anti-inflammatory function of PGRN in rheumatoid arthritis encouraged us to examine the role of PGRN in acute kidney injury (AKI). We found that levels of PGRN were significantly reduced in the kidney in a mouse model of renal ischemia/reperfusion injury. We also observed that PGRN deficiency (Grn(-/-) mice) significantly aggravated renal injury as evidenced by higher serum creatinine, more severe morphological injury, increased tubular epithelial cell death, and tubulointerstitial neutrophil and macrophage infiltration versus wild-type mice. In vitro, we found that recombinant human PGRN attenuated hypoxia-induced inflammatory actions and apoptosis in proximal tubule epithelial cells, at least in part associated with a nucleotide-binding oligomerization domain containing 2 (NOD2)-mediated immune response. Importantly, pretreatment with or delayed administration of recombinant human PGRN protected against or promoted recovery from renal ischemia/reperfusion injury in wild-type and Grn(-/-) mice. Similar protective effects were also found in cisplatin-induced AKI. Thus, our findings provide a better understanding of the biological activities of PGRN in the kidney and suggest that PGRN may be an innovative therapeutic strategy for treating patients with AKI.