Progression Of Tubulointerstitial Injury Research Articles

ATF5 regulates tubulointerstitial injury in diabetic kidney disease via mitochondrial unfolded protein response

BackgroundMitochondrial quality control (MQC) plays a critical role in the progression of tubulointerstitial injury in diabetic kidney disease (DKD). The mitochondrial unfolded protein response (UPRmt), which is an important MQC process, is activated to maintain mitochondrial protein homeostasis in response to mitochondrial stress. Activating transcription factor 5 (ATF5) is critical in the mammalian UPRmt via mitochondria-nuclear translocation. However, the role of ATF5 and UPRmt in tubular injury under DKD conditions is unknown.MethodsATF5 and UPRmt-related proteins including heat shock protein 60 (HSP60) and Lon peptidase 1 (LONP1), in DKD patients and db/db mice were examined by immunohistochemistry (IHC) and western blot analysis. Eight-week-old db/db mice were injected with ATF5-shRNA lentiviruses via the tail vein, and a negative lentivirus was used as a control. The mice were euthanized at 12 weeks, and dihydroethidium (DHE) and TdT-mediated dUTP nick end labeling (TUNEL) assays were performed to evaluate reactive oxygen species (ROS) production and apoptosis in kidney sections, respectively. In vitro, ATF5-siRNA, ATF5 overexpression plasmids or HSP60-siRNA were transfected into HK-2 cells to evaluate the effect of ATF5 and HSP60 on tubular injury under ambient hyperglycemic conditions. Mitochondrial superoxide (MitoSOX) staining was used to gauge mitochondrial oxidative stress levels, and the early stage of cell apoptosis was examined by Annexin V-FITC kits.ResultsIncreased ATF5, HSP60 and LONP1 expression was observed in the kidney tissue of DKD patients and db/db mice and was tightly correlated with tubular damage. The inhibition of HSP60 and LONP1, improvements in serum creatinine, tubulointerstitial fibrosis and apoptosis were observed in db/db mice treated with lentiviruses carrying ATF5 shRNA. In vitro, the expression of ATF5 was increased in HK-2 cells exposed to high glucose (HG) in a time-dependent manner, which was accompanied by the overexpression of HSP60, fibronectin (FN) and cleaved-caspase3 (C-CAS3). ATF5-siRNA transfection inhibited the expression of HSP60 and LONP1, which was accompanied by reduced oxidative stress and apoptosis in HK-2 cells exposed to sustained exogenous high glucose. ATF5 overexpression exacerbated these impairments. HSP60-siRNA transfection blocked the effect of ATF5 on HK-2 cells exposed to continuous HG treatment. Interestingly, ATF5 inhibition exacerbated mitochondrial ROS levels and apoptosis in HK-2 cells in the early period of HG intervention (6 h).ConclusionsATF5 could exert a protective effect in a very early stage but promoted tubulointerstitial injury by regulating HSP60 and the UPRmt pathway under DKD conditions, providing a potential target for the prevention of DKD progression.

Kidney tubule iron loading in experimental focal segmental glomerulosclerosis

Kidney iron deposition may play a role in the progression of tubulointerstitial injury during chronic kidney disease. Here, we studied the molecular mechanisms of kidney iron loading in experimental focal segmental glomerulosclerosis (FSGS) and investigated the effect of iron-reducing interventions on disease progression. Thy-1.1 mice were injected with anti-Thy-1.1 monoclonal antibody (mAb) to induce proteinuria. Urine, blood and tissue were collected at day (D)1, D5, D8, D15 and D22 after mAb injection. Thy-1.1 mice were subjected to captopril (CA), iron-deficient (ID) diet or iron chelation (deferoxamine; DFO). MAb injection resulted in significant albuminuria at all time points (p < 0.01). Kidney iron loading, predominantly in distal tubules, increased in time, along with urinary kidney injury molecule-1 and 24p3 concentration, as well as kidney mRNA expression of Interleukin-6 (Il-6) and Heme oxygenase-1 (Ho-1). Treatment with CA, ID diet or DFO significantly reduced kidney iron deposition at D8 and D22 (p < 0.001) and fibrosis at D22 (p < 0.05), but not kidney Il-6. ID treatment increased kidney Ho-1 (p < 0.001). In conclusion, kidney iron accumulation coincides with progression of tubulointerstitial injury in this model of FSGS. Reduction of iron loading halts disease progression. However, targeted approaches to prevent excessive kidney iron loading are warranted to maintain the delicate systemic and cellular iron balance.

Connectivity mapping of a chronic kidney disease progression signature identified lysine deacetylases as novel therapeutic targets

Tubulointerstitial injury is an important determinant of chronic kidney disease progression, yet treatment is limited. Accordingly, we derived a chronic kidney disease progression signature based on aging and disease in Col4a3-/- mice, a model associated with proteinuria and progressive loss of kidney function. Computational drug repurposing with the Connectivity Map identified vorinostat, a lysine deacetylase inhibitor, as a candidate treatment to reverse progression signature gene expression. Vorinostat administration significantly increased the lifespan of Col4a3-/- mice and attenuated tubulointerstitial fibrosis and JNK phosphorylation in the kidneys of Col4a3-/- mice. Invitro, vorinostat reduced albumin- and angiotensin II-induced activation of canonical mitogen-activated protein kinases in kidney tubular epithelial cells. Finally, a subset of murine progression signature genes was differentially expressed across kidney transcriptomic data from patients with focal segmental glomerulosclerosis, IgA nephropathy, and diabetic nephropathy. Thus, our findings suggest that lysine deacetylase inhibition may be a novel treatment to chronic kidney disease associated with proteinuria and progressive tubulointerstitial injury.

MON-023 HYPOKALEMIC NEPHROPATHY IN NEPHROTIC SYNDROME PATIENT

Hypokalemic nephropathy (HN) caused by prolonged K+ deficiency is associated with metabolic alkalosis, polydipsia, polyuria, growth retardation, hypertension, and progressive tubulointerstitial injury. Histopahologic report of HN in nephrotic syndrome (NS) patient is rare. A 27 year male suffered from weakness, especially lower leg, since two weeks prior to admission. The patients had a history of NS since childhood and cared by nephrologist. Recent condition was hemodynamically stable, laboratory examination showed potassium serum 1.85 mmol/L, albumin plasma 1.8 g/dL, serum creatinine 2.45 mg/dL, and marked dyslipidemia. The patient was clinically assesed with hypokalemia, relapse nephrotic syndrome and renal insufficiency. Intravenous potassium was given and renal biopsy was performed. Histopathologic findings showed global sclerosis. The specific findings may related to HN were mesangial proliferation and specific lesions on tubules including athrophy, vacuolization, intratubular deposition of amorphous and laminated hyaline materials and colloidisation of tubular epithelial, and interstitial nephritis. The patient was discharged at 10th day of hospitalization with potassium level 2.93 mmol/L, and serum creatinine 1.95 mg/dL. The patient was treated with oral methylprednisolone, mychophenolate mofetil, ACE-inhibitor, simvastatin and oral potassium Hypokalemic nephropathy may associate with chronic hypokalemia. Hypokalemia, especially if persistently occurs, can induce a variety of changes in renal tissue, impairing tubular transport and possibly inducing chronic tubulointerstitial disease and cyst formation. HN also associated with alterations in intrarenal vasoactive substances, leading to vasoconstriction, salt-sensitivity, and progression of interstitial fibrosis. Histopathological changes in HN, including severe tubular dilatation, intratubular deposition of amorphous and laminated hyaline materials, intratubular cellular casts, and tubular atrophy is found. More attention is paid to athrophy and vacuolar changes in renal tubular epithelium accompanied by inflammatory interstitial changes in patients with potassium losses. One function that is not impaired is the ability to appropriately conserve potassium, which can be important in distinguishing between extrarenal and renal sources of potassium losses when the cause of hypokalemia is not clear.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) A case with nephrotic syndrome associated with HN is reported. Chronic hypokalemia may involve in the development of hypokalemic nephropathy. Hallmark of histopathologic finding are tubular epithel athrophy, intratubular amorphous deposition and vacuolization of tubular cells, and interstitial nephritis to fibrosis.

Noninvasive Identification of Renal Hypoxia in Experimental Myocardial Infarctions of Different Sizes by Using BOLD MR Imaging in a Mouse Model.

Purpose To test the feasibility of using blood oxygen level-dependent (BOLD) magnetic resonance (MR) imaging to measure alterations in renal oxygenation in a mouse model with experimental myocardial infarctions (MIs) of different sizes. Materials and Methods The study was approved by the local animal ethics committee. One hundred eighty-nine male C57BL/6 J mice were randomly subjected to MI surgery (with different locations of left anterior descending coronary artery occlusion) or sham surgery, defined as the exposure of the heart but no ligation. Mice with MI underwent late gadolinium enhancement imaging 1 day after occlusion to confirm infarct size. Mice were sorted into three groups: those with large MI (n = 48), those with small MI (n = 48), and those with sham operation (n = 36). Renal BOLD MR imaging was performed before and 1, 7, 14, 28, and 60 days after MI, and histologic analysis of renal hypoxia-inducible factor-1α (HIF-1α) and kidney injury molecule-1 (KIM-1) was performed to evaluate tissue hypoxia and kidney injury in subgroups imaged at each time point. The relationships between the BOLD R2* and HIF-1α expression and between HIF-1α and KIM-1 expression were assessed. Statistical analyses were performed with one-way analysis of variance or the Kruskal-Wallis test and Spearman correlation test. Results A significant elevation in R2* was detected in the MI groups compared with the sham group in the cortex (P < .001 for large MI vs sham group; P = .007 for small MI vs sham group) and medulla (P < .001 for large MI vs sham group; P = .003 for small MI vs sham group) on day 60, and R2* was higher in the large MI group than in the small MI group (P < .001). Renal HIF-1α expression was increased after MI and showed linear correlation with R2* in the cortex (R2 = 0.56) and medulla (R2 = 0.63). In addition, an increase in renal KIM-1 was observed in the MI groups compared with the sham group on day 60 (sham group, 53.9 × 103 arbitrary units [au] ± 35.2; large MI group, 389.3 × 103 au ± 99.8; and small MI group, 185.8 × 103 au ± 91.9; P < .001 for large MI group vs sham group; P = .037 for small MI group vs sham group), and renal KIM-1 showed a positive correlation with HIF-1α (R2 = 0.68). Conclusion The magnitude of renal hypoxia with MIs of different sizes can be noninvasively measured with BOLD MR imaging, and increased renal hypoxia is a potential risk factor for progressive tubulointerstitial injury in mouse kidneys. © RSNA, 2017 Online supplemental material is available for this article.

Protective effect of nitric oxide in aristolochic acid-induced toxic acute kidney injury: an old friend with new assets.

Aristolochic acid (AA) nephropathy (AAN), a progressive tubulointerstitial injury of toxic origin, is characterized by early and transient acute tubular necrosis. This process has been demonstrated to be associated with reduced nitric oxide (NO) production, which can disrupt the regulation of renal function. In this study, we tested the hypothesis that L-arginine (L-Arg) supplementation could restore renal function and reduce renal injury after AA intoxication. C57BL/6 J male mice were randomly subjected to daily i.p. injection of either sterile saline solution or AA (2.5 mg kg(-1)) for 4 days. To determine whether AA-induced renal injuries were linked to reduced NO production, L-Arg, a substrate for NO synthase, was supplemented (5%) in drinking water. Mice intoxicated with AA exhibited features of rapid-onset acute kidney injury, including polyuria, significantly increased plasma creatinine concentrations, proteinuria and fractional excretion of sodium (P < 0.05), along with severe proximal tubular cell injury and increased NADPH oxidase 2 (Nox2)-derived oxidative stress (P < 0.05). This was associated with a significant reduction in NO bioavailability. L-Arg supplementation in AA-treated mice significantly increased NO bioavailability, which in turn improved renal function (creatininaemia, polyuria, proteinuria, fractional excreted sodium and N-acetyl-β-D-glucosaminidase enzymuria) and renal structure (tubular necrosis and tubular cell apoptosis). These changes were associated with significant reductions in Nox2 expression and in production of reactive oxygen species and with an increase in antioxidant concentrations. Our results demonstrate that preservation of NO bioavailability leads to renal protection in AA-induced acute kidney injury by reducing oxidative stress and maintaining renal function.

Hypoxia decreases podocyte expression of slit diaphragm proteins

BackgroundChronic hypoxia contributes to progressive tubulointerstitial injury and, consequently, renal failure. However, the effect of hypoxia on glomerular podocytes, which are integral to the slit diaphragm complex and responsible for selectivity of the glomerular filtration barrier, has not been completely determined.MethodsConditionally immortalized mouse podocyte cells were exposed to hypoxic (1% O2) or normoxic (room air) conditions for 24, 48, or 72 hours, after which cell viability was determined by MTT assay. Cells were stained with podocin and phalloidin to determine podocin and intracellular actin distribution. Expression of synaptopodin, CD2-associated protein (CD2AP), NcK, transforming growth factor-β1 (TGF-β1), hypoxia-inducible factor (HIF-1α) were evaluated by real-time polymerase chain reaction.ResultsPodocytes exposed to hypoxia had significantly reduced viability at 48 (87%) and 72 hours (66%). There was disarrangement of intracellular filament actin by phalloidin staining, a 30% weaker fluorescence intensity by podocin staining, significantly reduced expression of synaptopodin (12%), CD2AP (42%), NcK (38%), and increased expression of TGF-β1 and P-ERK after hypoxia treatment.ConclusionPodocyte exposure to hypoxia leads to reduced viability and SD protein expression, which may explain persistent and/or increasing proteinuria in patients with progressive renal failure. Increased expression of TGF-β1 and P-ERK is associated with apoptosis and fibrosis, which could be the link between hypoxia and glomerular injury.

Eicosapentaenoic acid regulates IκBα and prevents tubulointerstitial injury in kidney

Fish oil containing n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is well known to prevent the progression of IgA nephropathy. However, the mechanism through which fish oil prevents the progression of renal injury remains uncertain. We tried to clarify the effects of EPA on tubulointerstitial injury in the kidney both in vivo and in vitro. We examined the effects of EPA, especially to focus on nuclear factor kappa B (NF-κB), using Thy-1 nephritis models. Also the mechanism of EPA was investigated using small-interfering RNA (siRNA) in lipopolysaccharide (LPS)-stimulated proximal tubular epithelial cells (PTECs). In Thy-1 nephritis models, EPA significantly inhibited tubulointerstitial injury and the infiltration of macrophages into tubulointerstitial lesions except severe glomerular injury at early stage. Compared with control animals, NF-κB activation was significantly augmented in the Thy-1 nephritic kidney. However, treatment with EPA significantly reduced NF-κB activation, down-regulated the expressions of NF-κB-dependent molecules. Also in LPS-stimulated PTECs, LPS augmented NF-κB activation and the expression of NF-κB-dependent molecules. As in the case with the Thy-1 nephritis models, treatment with EPA inhibited them, prevented the degradation of IκBα in LPS-stimulated PTECs. Pre-treatment with siRNA for IκBα abolished the inhibitory effect of EPA on LPS-induced NF-κB activation, suggesting that EPA inhibited NF-κB activation by regulating IκBα. Our results indicate that EPA prevents the early progression of tubulointerstitial injury in Thy-1 nephritis models, and the inhibitory effect of EPA on the expression of inflammatory molecules via the regulation of IκBα in cultured cells may explain this mechanism.

Nestin is a novel marker for renal tubulointerstitial injury in immunoglobulin A nephropathy

Nestin, an intermediate filament originally identified as a marker of neural progenitor cells, is transiently expressed in endothelial cells and tubuloepithelial cells during kidney development. However, in adult kidneys, podocytes are the only cells that express nestin. In this study, we examined tubulointerstitial nestin expression in human glomerulonephritis. Renal biopsy specimens obtained from 41 adult patients with immunoglobulin (Ig)A nephropathy were studied. Nestin expression was determined by immunohistochemical staining and estimated by digital image analysis. To identify the phenotype of nestin-positive cells, a double immunofluorescent study was performed for nestin and CD34 (a marker for endothelial cells) or alpha-smooth muscle actin (alpha-SMA, a marker for myofibroblasts). In normal kidney, nestin expression was restricted to the podocytes and was not detected in tubular cells and tubulointerstitial cells. In contrast, increased nestin expression was observed at tubulointerstitial areas of IgA nephropathy. The degree of tubulointerstitial nestin expression was positively correlated with tubulointerstitial fibrosis (r = 0.546, P < 0.001). The double immunofluorescent study showed that most nestin-positive cells in the interstitium were co-stained with CD34 or alpha-SMA, suggesting that peritubular endothelial cells and tubulointerstitial myofibroblasts express nestin during the progression of tubulointerstitial injury. In addition, strong nestin expression was associated with deterioration of renal function. Nestin expression is associated with tubulointerstitial injury and predicts renal prognosis in IgA nephropathy. Nestin could be a new marker for peritubular endothelial cell injury and tubulointerstitial fibrosis.

Amelioration of metabolic acidosis in patients with low GFR reduced kidney endothelin production and kidney injury, and better preserved GFR

Metabolic acidosis often accompanies low glomerular filtration rate and induces secretion of endothelin, which in turn might mediate kidney injury. Here we tested whether treatment of metabolic acidosis in patients with low glomerular filtration rate reduced the progression of kidney disease. Fifty-nine patients with hypertensive nephropathy and metabolic acidosis had their blood pressure reduced with regimens that included angiotensin-converting enzyme inhibition. Thirty patients were then prescribed sodium citrate, and the remaining 29, unable or unwilling to take sodium citrate, served as controls. All were followed for 24 months with maintenance of their blood pressure reduction. Urine endothelin-1 excretion, a surrogate of kidney endothelin production, and N-acetyl-beta-D-glucosaminidase, a marker of kidney tubulointerstitial injury, were each significantly lower, while the rate of estimated glomerular filtration rate decline was significantly slower. The estimated glomerular filtration rate was statistically higher after 24 months of sodium citrate treatment compared to the control group. Hence it appears that sodium citrate is an effective kidney-protective adjunct to blood pressure reduction and angiotensin-converting enzyme inhibition.

Amitriptyline attenuates interstitial inflammation and ameliorates the progression of renal fibrosis

Amitriptyline is a pleiotropic tricyclic antidepressant, which has anti-oxidant and anti-inflammatory properties. We tested whether amitriptyline might be useful in the treatment of chronic renal disease using the mouse model of unilateral ureteral obstruction. Amitriptyline caused a significant reduction of interstitial fibrosis, determined by Masson's staining, with minimal myofibroblast formation and macrophage infiltration following ureteral obstruction. Using quantitative PCR we found that this treatment significantly reduced the expression of key molecular markers of progressive tubulointerstitial injury such as osteopontin, MCP-1, ICAM-1, and TGF-beta1 compared to their level in a saline-treated control group. Sublethal X-irradiation or mycophenolate mofetil, treatments that reduce inflammation, were comparable to amitriptyline in the reduction of interstitial fibrosis and macrophage infiltration. These studies in animals suggest that amitriptyline is worth testing as a therapeutic agent that might preserve renal function by blocking inflammation and renal fibrosis.

Osmotic polyuria: an overlooked mechanism in diabetic nephropathy

Tubulo-interstitial pathology in diabetic nephropathy is thought to be caused by cell injury that is induced by high ambient glucose levels and increased proportions of glycated proteins. Other mechanistic hypotheses engage glomerular ultrafiltration of proteins and bioactive growth factors and their effects on tubular cells. Some scholars promote tubular ischaemia due to reduced peritubular blood flow as a response to glomerular injury. All of these mechanisms contribute to renal tubulo-interstitial injury in diabetic nephropathy. However, they do not well explain observations that have been made in studies of experimental animals and evaluations of human biopsies showing dilated collecting ducts in early diabetic nephropathy. Dilatation of distal nephron segments is routinely seen in human biopsies or in histological sections from experimental diabetic nephropathy and is reminiscent of similar findings in obstructive nephropathy. Moreover, it is these dilated tubules that are the primary source for pro-inflammatory and pro-fibrogenic cytokines and regulators. Based on this large body of observations from this laboratory and the published literature this narrative develops a novel hypothesis where hyperglycaemic, osmotic polyuria play important contributory roles in the initiation and progression of tubulo-interstitial injury in diabetic nephropathy.

NF-κB-dependent genes induced by proteinuria and identified using DNA microarrays

A close correlation has been shown between tubulointerstitial (TI) injury and the outcome of renal dysfunction, and nuclear factor-kappaB (NFkappaB) has been shown to play a key role in proteinuria-induced TI injury. To explore the molecular mechanisms of the proteinuria-induced TI injury further, we have analyzed renal gene expression with DNA microarrays, with and without specific inhibition of NF-kappaB in the proximal tubules. Unilaterally nephrectomized rats loaded with bovine serum albumin (BSA) were used as a model of proteinuric renal injury. Renal NF-kappaB activation was inhibited by gene transfer of the truncated form of IkappaBalpha (inhibitor of NF-kappaB) via the injection of a recombinant adenovirus vector into the renal artery, an method established in a previous study. Total RNA was extracted from the kidney and analyzed with a DNA microarrays containing 1081 genes. Renal NF-kappaB activation and TI injury in BSA-loaded proteinuric rats were inhibited by the gene transfer of the truncated form of IkappaBalpha. DNA microarray analysis revealed 45 up-regulated genes and six down-regulated genes in the proteinuric rats, and expression of 23 of these 51 genes was significantly altered by NF-kappaB inhibition. Among these 23 genes, we focused on clusterin and confirmed the results of microarray analysis by Western blotting and PCR. In this study, 23 genes of 51 proteinuria-related genes were regulated by NF-kappaB activation, suggesting that some of these genes may serve as target molecules for the treatment of progressive TI injury.

Peritubular Ischemia Contributes More to Tubular Damage than Proteinuria in Immune-Mediated Glomerulonephritis

Proteinuria is a key factor in the progression of tubulointerstitial injury. Recently, tubular ischemia as a result of loss of peritubular capillaries has been identified as another major contributor to disease progression, but the relative contribution of these insults on tubulointerstitial damage

The alternative pathway of complement is activated in the glomeruli and tubulointerstitium of mice with adriamycin nephropathy

The complement system effectively identifies and clears invasive pathogens as well as injured host cells. Uncontrolled complement activation can also contribute to tissue injury, however, and inhibition of this system may ameliorate many types of inflammatory injury. Several studies have demonstrated that the filtration of complement proteins into the renal tubules, as occurs during proteinuric renal disease, causes tubular inflammation and injury. In the present study, we tested the hypothesis that activation of the complement system in the urinary space requires an intact alternative pathway. Using a model of adriamycin-induced renal injury, which induces injury resembling focal segmental glomerulosclerosis, we examined whether mice deficient in factor B would be protected from the development of progressive tubulointerstitial injury. Complement activation was attenuated in the glomeruli and tubulointerstitium of mice with congenital deficiency of factor B (fB-/-) compared with wild-type controls, demonstrating that complement activation does occur through the alternative pathway. Deficiency in factor B did not significantly protect the mice from tubulointerstitial injury. However, treatment of wild-type mice with an inhibitory monoclonal antibody to factor B did delay the development of renal failure. These results demonstrate that complement activation in this nonimmune complex-mediated model of progressive renal disease requires an intact alternative pathway.

Proteomic identification of alterations in metabolic enzymes and signaling proteins in hypokalemic nephropathy

Hypokalemic nephropathy caused by prolonged K(+) deficiency is associated with metabolic alkalosis, polydipsia, polyuria, growth retardation, hypertension, and progressive tubulointerstitial injury. Its pathophysiology, however, remains unclear. We performed gel-based, differential proteomics analysis of kidneys from BALB/c mice fed with high-normal-K(+) (HNK), low-normal-K(+) (LNK), or K(+)-depleted diet for 8 wk (n = 6 in each group). Plasma K(+) levels were 4.62 +/- 0.35, 4.46 +/- 0.23, and 1.51 +/- 0.21 mmol/L for HNK, LNK, and KD mice, respectively (p < 0.0001; KD vs. others). With comparable amounts of food intake, the KD mice drank significantly more water than the other two groups and had polyuria. Additionally, the KD mice had growth retardation, metabolic alkalosis, markedly enlarged kidneys, renal tubular dilation, intratubular deposition of amorphous and laminated hyaline materials, and tubular atrophy. A total of 33 renal proteins were differentially expressed between the KD mice and others, whereas only eight proteins were differentially expressed between the HNK and LNK groups, as determined by quantitative intensity analysis and ANOVA with Tukey's post hoc multiple comparisons. Using MALDI-MS and/or quadrupole-TOF MS/MS, 30 altered proteins induced by K(+)-depletion were identified as metabolic enzymes (e.g., carbonic anhydrase II, aldose reductase, glutathione S-transferase GT41A, etc.), signaling proteins (14-3-3 epsilon, 14-3-3 zeta, and cofilin 1), and cytoskeletal proteins (gamma-actin and tropomyosin). Some of these altered proteins, particularly metabolic enzymes and signaling proteins, have been demonstrated to be involved in metabolic alkalosis, polyuria, and renal tubular injury. Our findings may lead to a new road map for research on hypokalemic nephropathy and to better understanding of the pathophysiology of this medical disease when the functional and physiological significances of these altered proteins are defined.

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

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. 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. 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. Maneuvers to activate HIF in the ischemic tubulointerstitium will be a new direction to future therapeutic strategies.

Hypoperfusion of peritubular capillaries induces chronic hypoxia before progression of tubulointerstitial injury in a progressive model of rat glomerulonephritis.

Chronic hypoxia likely plays a pivotal role in chronic renal disease, but the specifics of its involvement remain unclear. To elucidate how chronic hypoxia occurs and whether hypoxia participates in the progression of renal disease, the authors established an irreversible glomerulonephritis model induced by uninephrectomy and repeated anti-Thy-1 antibody injections. Glomerulosclerosis with microvascular obliteration was complete at 2 wk after antibody injection and was not restored until 11 wk. Tubulointerstitial injury was mild at 2 wk and was gradually exacerbated until 11 wk, a pattern that was in accordance with the loss of peritubular capillaries. Immunohistochemical analysis using pimonidazole revealed the augmentation of hypoxia in the cortex before the aggravation of tubulointerstitial injury and subsequent peritubular capillary loss. The preexistence of hypoxia implies that it had substantial participation in the progression of tubulointerstitial injury. To test whether blood flow was inhibited in diseased kidneys, capillaries with intact blood flow were identified by tail vein injection of biotinylated lectin specific to endothelial cells. The renal microvasculature was well recognized by lectin in the controls, whereas lectin binding to peritubular capillaries was strikingly decreased in diseased kidneys, suggesting a disturbance of blood flow. Intravital microscopy analysis confirmed that blood flow in peritubular capillaries was decreased by approximately 40% in the disease group compared with the controls. In conclusion, stagnation of blood flow in peritubular capillaries induced chronic hypoxia at an early stage in this model, which was followed by progressive tubulointerstitial injury and a loss of peritubular capillaries.

Obstructive uropathy in mice and humans: potential role for PDGF-D in the progression of tubulointerstitial injury.

Tubulointerstitial fibrosis is a major characteristic of progressive renal diseases. Platelet-derived growth factor (PDGF) is a family of growth regulatory molecules consisting of PDGF-A and -B, along with the newly discovered PDGF-C and -D. They signal through cell membrane receptors, PDGF receptor alpha (PDGF-Ralpha) and receptor beta (PDGF-Rbeta). Involvement of PDGF-B and PDGF-Rbeta in the initiation and progression of renal fibrosis has been well documented. The authors studied the localization of PDGF ligands and receptors by immunohistochemistry, with emphasis on the role of PDGF-D in murine renal fibrosis induced by unilateral ureteral obstruction (UUO). In mice with UUO, de novo expression of PDGF-D was detected in interstitial cells at day 4, which increased to maximal expression at day 14. Increased expression of PDGF-B by interstitial cells and in some tubules was observed after day 4. The diseased mice did not show augmentation of PDGF-A or PDGF-C proteins in the areas of fibrosis. PDGF-Ralpha and -Rbeta protein expression was increased in interstitial cells after day 4 and reached maximal expression at day 14. Human renal nephrectomies (n = 10) of chronic obstructive nephropathy demonstrated similar de novo expression of PDGF-D in interstitial cells, correlating with expression of PDGF-Rbeta and PDGF-B, as it did in the murine model. These observations suggest that PDGF-D plays an important role in the pathogenesis of tubulointerstitial injury through binding of PDGF-Rbeta in both human obstructive nephropathy and the corresponding murine model of UUO.

Progression of tubulointerstitial injury by osteopontin-induced macrophage recruitment in advanced diabetic nephropathy of transgenic (mRen-2)27 rats.

Osteopontin is a macrophage chemotactic protein that has been pathogenetically linked to tissue injury in non-diabetic kidney disease. To examine osteopontin expression and macrophage accumulation in diabetic nephropathy, diabetes was induced with streptozotocin (STZ) in the transgenic (mRen-2)27 rat, a rodent model which develops the structural and functional features of its human counterpart when rendered diabetic. Non-diabetic rats were randomly selected to receive either (STZ) or citrate buffer. Diabetic rats were further randomly selected to receive either the angiotensin-converting-enzyme inhibitor, perindopril (6 mg/kg/day), or the vehicle only for 12 weeks. When compared with control animals, diabetes was associated with a 10-fold increase in the gene expression of osteopontin. Increased transcript and immunostainable osteopontin were detected in tubular epithelial cells in association with extensive macrophage accumulation. Treatment with perindopril significantly ameliorated the overexpression of osteopontin in association with attenuation of macrophage accumulation. These findings suggest that osteopontin expression and macrophage accumulation may play a role in the tubulointerstitial injury in diabetic nephropathy, and that inhibition of osteopontin expression may be one of the mechanisms by which blockade of the renin-angiotensin system confers a renoprotective effect.