Podocyte-Specific Deletion of MCP-1 Fails to Protect against Angiotensin II- or Adriamycin-Induced Glomerular Disease.

Investigating the role of podocytes in proteinuric disease is imperative to address the increasing global burden of chronic kidney disease (CKD). Studies strongly implicate increased levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) in proteinuric CKD. Since podocytes express the receptor for MCP-1 (i.e., CCR2), we hypothesized that podocyte-specific MCP-1 production in response to stimuli could activate its receptor in an autocrine manner, leading to further podocyte injury. To test this hypothesis, we generated podocyte-specific MCP-1 knockout mice (Podo-Mcp-1fl/fl) and exposed them to proteinuric injury induced by either angiotensin II (Ang II; 1.5 mg/kg/d, osmotic minipump) or Adriamycin (Adr; 18 mg/kg, intravenous bolus). At baseline, there were no between-group differences in body weight, histology, albuminuria, and podocyte markers. After 28 days, there were no between-group differences in survival, change in body weight, albuminuria, kidney function, glomerular injury, and tubulointerstitial fibrosis. The lack of protection in the knockout mice suggests that podocyte-specific MCP-1 production is not a major contributor to either Ang II- or Adr-induced glomerular disease, implicating that another cell type is the source of pathogenic MCP-1 production in CKD.

NRF2 in kidney physiology and disease.

The role of NRF2 in kidney biology has received considerable interest over the past decade. NRF2 transcriptionally controls genes responsible for cellular protection against oxidative and electrophilic stress and has anti-inflammatory functions. NRF2 is expressed throughout the kidney and plays a role in salt and water handling. In disease, animal studies show that NRF2 protects against tubulointerstitial damage and reduces interstitial fibrosis and tubular atrophy, and may slow progression of polycystic kidney disease. However, the role of NRF2 in proteinuric glomerular diseases is controversial. Although the NRF2 inducer, bardoxolone methyl (CDDO-Me), increases glomerular filtration rate in humans, it has not been shown to slow disease progression in diabetic kidney disease and Alport syndrome. Furthermore, bardoxolone methyl was associated with negative effects on fluid retention, proteinuria, and blood pressure. Several animal studies replicate findings of worsened proteinuria and a more rapid progression of kidney disease, although considerable controversy exists. It is clear that further study is needed to better understand the effects of NRF2 in the kidney. This review summarizes the available data to clarify the promise and risks associated with targeting NRF2 activity in the kidney.

Suppression of NRF2 Activity by HIF-1α Promotes Fibrosis after Ischemic Acute Kidney Injury.

Acute kidney injury (AKI) is a rapid decline in renal function and can occur after ischemia/reperfusion injury (IRI) to the tubular epithelia. The nuclear factor erythroid-2-related factor 2 (NRF2) pathway protects against AKI and AKI-to-chronic kidney disease (CKD) progression, but we previously demonstrated that severe IRI maladaptively reduced NRF2 activity in mice. To understand the mechanism of this response, we subjected C57BL/6J mice to unilateral kidney IRI with ischemia times that were titrated to induce mild to severe injury. Mild IRI increased NRF2 activity and was associated with renal recovery, whereas severe IRI decreased NRF2 activity and led to progressive CKD. Due to these effects of ischemia, we tested the hypothesis that hypoxia-inducible factor-1α (HIF-1α) mediates NRF2 activity. To mimic mild and severe ischemia, we activated HIF-1α in HK-2 cells in nutrient-replete or nutrient-deficient conditions. HIF-1α activation in nutrient-replete conditions enhanced NRF2 nuclear localization and activity. However, in nutrient-deficient conditions, HIF-1α activation suppressed NRF2 nuclear localization and activity. Nuclear localization was rescued with HIF-1α siRNA knockdown. Our results suggest that severe ischemic AKI leads to HIF-1α-mediated suppression of NRF2, leading to AKI-to-CKD progression.

Cubilin-, megalin-, and Dab2-dependent transcription revealed by CRISPR/Cas9 knockout in kidney proximal tubule cells.

The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.

Genetic or pharmacologic Nrf2 activation increases proteinuria in chronic kidney disease in mice.

The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway upregulates key cellular defenses. Clinical trials are utilizing pharmacologic Nrf2 inducers such as bardoxolone methyl to treat chronic kidney disease, but Nrf2 activation has been linked to a paradoxical increase in proteinuria. To understand this effect, we examined genetically engineered mice with elevated Nrf2 signaling due to reduced expression of the Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1). These Keap1FA/FA mice lacked baseline proteinuria but exhibited increased proteinuria in experimental models evoked by adriamycin, angiotensin II, or protein overload. After injury, Keap1FA/FA mice had increased glomerulosclerosis, nephrin disruption and shedding, podocyte injury, foot process effacement, and interstitial fibrosis. Keap1FA/FA mice also had higher daytime blood pressures and lower heart rates measured by radiotelemetry. Conversely, Nrf2 knockout mice were protected from proteinuria. We also examined the pharmacologic Nrf2 inducer CDDO-Im. Compared to angiotensin II alone, the combination of angiotensin II and CDDO-Im significantly increased proteinuria, a phenomenon not observed in Nrf2 knockout mice. This effect was not accompanied by additional increases in blood pressure. Finally, Nrf2 was found to be upregulated in the glomeruli of patients with focal segmental glomerulosclerosis, diabetic nephropathy, fibrillary glomerulonephritis, and membranous nephropathy. Thus, our studies demonstrate that Nrf2 induction in mice may exacerbate proteinuria in chronic kidney disease.

Co-administering Melatonin With an Estradiol-Progesterone Menopausal Hormone Therapy Represses Mammary Cancer Development in a Mouse Model of HER2-Positive Breast Cancer.

Melatonin has numerous anti-cancer properties reported to influence cancer initiation, promotion, and metastasis. With the need for effective hormone therapies (HT) to treat menopausal symptoms without increasing breast cancer risk, co-administration of nocturnal melatonin with a natural, low-dose HT was evaluated in mice that develop primary and metastatic mammary cancer. Individually, melatonin (MEL) and estradiol-progesterone therapy (EPT) did not significantly affect mammary cancer development through age 14 months, but, when combined, the melatonin-estradiol-progesterone therapy (MEPT) significantly repressed tumor formation. This repression was due to effects on tumor incidence, but not latency. These results demonstrate that melatonin and the HT cooperate to decrease the mammary cancer risk. Melatonin and EPT also cooperate to alter the balance of the progesterone receptor (PR) isoforms by significantly increasing PRA protein expression only in MEPT mammary glands. Melatonin significantly suppressed amphiregulin transcripts in MEL and MEPT mammary glands, suggesting that amphiregulin together with the higher PRA:PRB balance and other factors may contribute to reducing cancer development in MEPT mice. Melatonin supplementation influenced mammary morphology by increasing tertiary branching in the mouse mammary glands and differentiation in human mammary epithelial cell cultures. Uterine weight in the luteal phase was elevated after long-term exposure to EPT, but not to MEPT, indicating that melatonin supplementation may reduce estrogen-induced uterine stimulation. Melatonin supplementation significantly decreased the incidence of grossly-detected lung metastases in MEL mice, suggesting that melatonin delays the formation of metastatic lesions and/or decreases aggressiveness in this model of HER2+ breast cancer. Mammary tumor development was similar in EPT and MEPT mice until age 8.6 months, but after 8.6 months, only MEPT continued to suppress cancer development. These data suggest that melatonin supplementation has a negligible effect in young MEPT mice, but is required in older mice to inhibit tumor formation. Since melatonin binding was significantly decreased in older mammary glands, irrespective of treatment, melatonin supplementation may overcome reduced melatonin responsiveness in the aged MEPT mice. Since melatonin levels are known to decline near menopause, nocturnal melatonin supplementation may also be needed in aging women to cooperate with HT to decrease breast cancer risk.

The Effect of Melatonin Upon Postacute Withdrawal Among Males in a Residential Treatment Program (M-PAWS): A Randomized, Double-blind, Placebo-controlled Trial.

OBJECTIVE: Assess the effect of melatonin (5 mg) compared with placebo as an adjuvant treatment along with current behavioral and pharmacotherapy for 28 days on weekly self-reported severity of anxiety, depression, stress, and sleep complaints, and also how sleep is affecting daily life in males 18 years of age and older in recovery from substance use at a residential program in south-western Pennsylvania. BACKGROUND: Individuals in recovery experience a variety of symptoms including, but are not limited to, anxiety, depression, sleep difficulties, and stress. In the U.S., melatonin is a readily available nutraceutical that is used to alleviate sleep difficulties. Studies also suggest that melatonin may also have anxiolytic and antidepressive actions alone, as well as in those with co-morbid insomnia. Observation of clinicians treating individuals during and/or post drug cessation indicated that melatonin is commonly provided specifically to alleviate sleep difficulties with little evidence regarding efficacy in this population. The paucity of evidence as well as observation of clinical practices provided the rationale for this randomized clinical trial. METHODS: A single-center, randomized, double-blind, placebo-controlled, parallel-group trial was conducted. Seventy individuals were enrolled, block-randomized with an allocation ratio of 1:1. Intention-to-treat analysis was performed for all primary outcome measures. Primary outcome measures were assessed with the Generalized Anxiety Disorder Scale (GAD-7), Personal Health Questionnaire Depression Scale (PHQ-8), Perceived Stress Scale (PSS-14), and Pittsburgh Sleep Symptom Questionnaire-Insomnia (PSSQ-1). Secondary outcome measures were to acquire participant characteristics, determine adherence, and document adverse events. RESULTS: No statistically significant between-group differences were detected for baseline characteristics. Even though the proportion of individuals reporting an adverse event between groups was not significantly different, the frequency of reported adverse events was greater in the melatonin group. Intention-to-treat analysis for all the measured outcomes revealed no statistically significant between-group differences for same day comparisons. CONCLUSIONS: The diversity of medication regimens, and also the services provided by the residential treatment site add to the complexity of assessing the efficacy of melatonin on the measured outcomes. Given these limitations, there exists insufficient evidence to suggest that the effect of melatonin and placebo on the outcomes were significantly different.

Effects on bone by the light/dark cycle and chronic treatment with melatonin and/or hormone replacement therapy in intact female mice.

In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV-Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd-/-) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real-time RT-PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17ß-estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.

NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts.

TGF-beta1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase-derived ROS in TGF-beta1-induced activation of rat kidney fibroblasts and expression of alpha-smooth muscle actin (alpha-SMA) and Fn-ED-A. In vitro, TGF-beta1 stimulated formation of abundant stress fibers and increased expression of both alpha-SMA and Fn-ED-A. In addition, TGF-beta1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-beta1-induced stimulation of NADPH oxidase activity and reduced alpha-SMA and Fn-ED-A expression. Inhibition of TGF-beta1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-beta1-enhanced NADPH oxidase activity; and decreased expression of Nox4, alpha-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-beta1-induced Smad3 but reduced both alpha-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked alpha-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-beta1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-beta1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased alpha-SMA and Fn-ED-A. Taken together, these results suggest that TGF-beta1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.

Recognition of benztropine by the dopamine transporter (DAT) differs from that of the classical dopamine uptake inhibitors cocaine, methylphenidate, and mazindol as a function of a DAT transmembrane 1 aspartic acid residue.

Binding of cocaine to the dopamine transporter (DAT) protein blocks synaptic dopamine clearance, triggering the psychoactive effects associated with the drug; the discrete drug-protein interactions, however, remain poorly understood. A longstanding postulate holds that cocaine inhibits DAT-mediated dopamine transport via competition with dopamine for formation of an ionic bond with the DAT transmembrane aspartic acid residue D79. In the present study, DAT mutations of this residue were generated and assayed for translocation of radiolabeled dopamine and binding of radiolabeled DAT inhibitors under identical conditions. When feasible, dopamine uptake inhibition potency and apparent binding affinity K(i) values were determined for structurally diverse DAT inhibitors. The glutamic acid substitution mutant (D79E) displayed values indistinguishable from wild-type DAT in both assays for the charge-neutral cocaine analog 8-oxa-norcocaine, a finding not supportive of the D79 "salt bridge" ligand-docking model. In addressing whether the D79 side chain contributes to the DAT binding sites of other portions of the cocaine pharmacophore, only inhibitors with modifications of the tropane ring C-3 substituent, i.e., benztropine and its analogs, displayed a substantially altered dopamine uptake inhibition potency as a function of the D79E mutation. A single conservative amino acid substitution thus differentiated structural requirements for benztropine function relative to those for all other classical DAT inhibitors. Distinguishing the precise mechanism of action of this DAT inhibitor with relatively low abuse liability from that of cocaine may be attainable using DAT mutagenesis and other structure-function studies, opening the door to rational design of therapeutic agents for cocaine abuse.