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1.  Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes 
PLoS ONE  2014;9(6):e99771.
Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin.
doi:10.1371/journal.pone.0099771
PMCID: PMC4055698  PMID: 24924335
2.  NPHS2 Variation in Sporadic Focal Segmental Glomerulosclerosis 
Mutations in NPHS2, the gene that encodes podocin, are well-established causes of both familial and sporadic steroid-resistant focal segmental glomerulosclerosis (FSGS) in the pediatric population, but have not been well-characterized in late-onset disease. To investigate the role of NPHS2 polymorphisms in sporadic cases of late-onset FSGS, we studied 377 biopsy-confirmed FSGS cases and 919 controls. We identified 18 single nucleotide polymorphisms (SNPs) by resequencing a subgroup of cases and controls, and subsequently genotyped African-American and European-American cases and controls for five missense SNPs, three SNPs within introns, and four SNPs in the 3′ untranslated region. No homozygotes or compound heterozygotes were observed for any missense mutation. R138Q carriers were more frequent among FSGS cases relative to controls (OR = 4.9, P = 0.06), but heterozygosity for the other four missense mutations was equally distributed among FSGS cases and controls. Finally, a common haplotype of noncoding SNPs carried by 20% of African-Americans, but not observed in European-Americans, was strongly associated with a 50% reduction in risk for sporadic FSGS (OR = 0.5, P = 0.001). These results indicate that genetic variation or mutation of NPHS2 may play a role in late-onset sporadic FSGS.
doi:10.1681/ASN.2007030319
PMCID: PMC4096868  PMID: 17942957
3.  Viruses and collapsing glomerulopathy: a brief critical review 
Clinical Kidney Journal  2012;6(1):1-5.
Background
Collapsing glomerulopathy may occur in an idiopathic (primary) form and in association with a wide spectrum of infectious and inflammatory conditions and medications. The association of collapsing glomerulopathy with human immunodeficiency virus (HIV)-1 infection is well established; less certain is the association with other viral infections.
Methods
We searched PubMed for articles in all languages that addressed glomerulopathies associated with parvovirus B19, cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis C virus (HCV) and simian virus 40 (SV40).
Results
Case reports and small-case series link infection with these common viruses and glomerular injury. The evidence for a pathogenic role is generally stronger for glomerulonephritis than for collapsing glomerulopathy.
Conclusions
The evidence linking collapsing glomerulopathy with CMV is relatively strong but not yet conclusive, while the evidence for a pathogenic role for EBV and parvovirus B19 is weaker.
doi:10.1093/ckj/sft002
PMCID: PMC3560379  PMID: 23372939
collapsing focal segmental glomerulosclerosis; cytomegalovirus; Epstein-Barr virus; parvovirus B19; podocyte
4.  APOL1 Risk Variants, Race, and Progression of Chronic Kidney Disease 
The New England journal of medicine  2013;369(23):2183-2196.
BACKGROUND
Among patients in the United States with chronic kidney disease, black patients are at increased risk for end-stage renal disease, as compared with white patients.
METHODS
In two studies, we examined the effects of variants in the gene encoding apolipoprotein L1 (APOL1) on the progression of chronic kidney disease. In the African American Study of Kidney Disease and Hypertension (AASK), we evaluated 693 black patients with chronic kidney disease attributed to hypertension. In the Chronic Renal Insufficiency Cohort (CRIC) study, we evaluated 2955 white patients and black patients with chronic kidney disease (46% of whom had diabetes) according to whether they had 2 copies of high-risk APOL1 variants (APOL1 high-risk group) or 0 or 1 copy (APOL1 low-risk group). In the AASK study, the primary outcome was a composite of end-stage renal disease or a doubling of the serum creatinine level. In the CRIC study, the primary outcomes were the slope in the estimated glomerular filtration rate (eGFR) and the composite of end-stage renal disease or a reduction of 50% in the eGFR from baseline.
RESULTS
In the AASK study, the primary outcome occurred in 58.1% of the patients in the APOL1 high-risk group and in 36.6% of those in the APOL1 low-risk group (hazard ratio in the high-risk group, 1.88; P<0.001). There was no interaction between APOL1 status and trial interventions or the presence of baseline proteinuria. In the CRIC study, black patients in the APOL1 high-risk group had a more rapid decline in the eGFR and a higher risk of the composite renal outcome than did white patients, among those with diabetes and those without diabetes (P<0.001 for all comparisons).
CONCLUSIONS
Renal risk variants in APOL1 were associated with the higher rates of end-stage renal disease and progression of chronic kidney disease that were observed in black patients as compared with white patients, regardless of diabetes status. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others.)
doi:10.1056/NEJMoa1310345
PMCID: PMC3969022  PMID: 24206458
5.  HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Reciprocal Effects on PPAR/GR Co-Regulation 
Science translational medicine  2013;5(213):213ra164.
Viral infections, such as HIV, have been linked to obesity, but mechanistic evidence that they cause adipose dysfunction in vivo is lacking. We investigated a pathogenic role for the HIV-1 accessory protein viral protein R (Vpr), which can coactivate the glucocorticoid receptor (GR) and co-repress peroxisome proliferator–activated receptor γ (PPARγ) in vitro, in HIV-associated adipose dysfunction. Vpr circulated in the blood of most HIV-infected patients tested, including those on antiretroviral therapy (ART) with undetectable viral load. Vpr-mediated mechanisms were dissected in vivo using mouse models expressing the Vpr transgene in adipose tissues and liver (Vpr-Tg) or infused with synthetic Vpr. Both models demonstrated accelerated whole-body lipolysis, hyperglycemia and hypertriglyceridemia, and tissue-specific findings. Fat depots in these mice had diminished mass, macrophage infiltration, and blunted PPARγ target gene expression but increased GR target gene expression. In liver, we observed blunted PPARα target gene expression, steatosis with decreased adenosine monophosphate– activated protein kinase activity, and insulin resistance. Similar to human HIV-infected patients, Vpr circulated in the serum of Vpr-Tg mice. Vpr blocked differentiation in preadipocytes through cell cycle arrest, whereas in mature adipocytes, it increased lipolysis with reciprocally altered association of PPARγ and GR with their target promoters. These results delineate a distinct pathogenic sequence: Vpr, released from HIV-1 in tissue reservoirs after ART, can disrupt PPAR/GR co-regulation and cell cycle control to produce adipose dysfunction and hepatosteatosis. Confirmation of these mechanisms in HIV patients could lead to targeted treatment of the metabolic complications with Vpr inhibitors, GR antagonists, or PPARγ/PPARα agonists.
doi:10.1126/scitranslmed.3007148
PMCID: PMC4009012  PMID: 24285483
6.  Tenofovir Treatment Duration Predicts Proteinuria in a Multi-Ethnic United States Cohort of Children and Adolescents with Perinatal HIV-1 Infection 
Background
Tenofovir is associated with renal proximal tubule injury. Such toxicity has not been extensively studied in HIV-1-infected children, in whom tenofovir is increasingly used.
Methods
History, urine and blood were collected at regular intervals from 448 children and adolescents with perinatal HIV-1 infection followed in the Pediatric HIV/AIDS Cohort study. Relationships between tenofovir use and proteinuria and chronic kidney disease (CKD) outcomes were examined using multivariable logistic regression models. Proteinuria was defined as at least one urine protein/creatinine ratio (uPCR) ≥0.2, and CKD as ≥2 sequential uPCR ≥0.2 or estimated glomerular filtration rates (eGFR) <60 mL/min/1.73 m2 with no subsequent resolution, or a clinical diagnosis not contradicted by a normal uPCR. Subjects with ≥2 uPCR <0.2, and no abnormal uPCR and eGFR comprised the comparison group.
Results
Subjects were 47% male, 72% black, 24% Hispanic, with entry mean age (±standard deviation) of 11.5±2.5 years. Proteinuria prevalence at entry, and annually during 3 years, ranged from 10.3%–13.7%. The cumulative prevalence of proteinuria was 22% (94/434, 95% CI: 18%–26%) and CKD 4.5% (20/448, 95% CI: 2.7%–6.8%). Duration of tenofovir use was an independent predictor of proteinuria, with >3 years of exposure having the highest risk compared with no exposure (OR: 2.53, 95% CI: 1.23- 5.22, overall p=0.01). Overall, duration of tenofovir use did not significantly predict the presence of CKD.
Conclusions
Rates of proteinuria and CKD were lower than those seen in the pre-HAART era. However, prolonged exposure to tenofovir increases risk of renal injury.
doi:10.1097/INF.0b013e31827f4eff
PMCID: PMC3800277  PMID: 23249917
Tenofovir; proteinuria; chronic kidney disease; proximal tubules; nephrotoxicity; urine protein/creatinine ratio
7.  Microalbuminuria in HIV Disease 
American journal of nephrology  2013;37(5):10.1159/000350384.
Background/Aims
Microalbuminuria is a marker for early kidney disease and cardiovascular risk. The purposes of this study were to determine the prevalence of microalbuminuria in an HIV-infected clinic population, to test the predictive value of a single urine albumin-creatinine ratio (ACR) to identify persistent microalbuminuria and to examine covariates of microalbuminuria.
Methods
We conducted a prospective cohort study of HIV-infected subjects (n=182) without proteinuria (P/C ratio ≥0.5 g/g), elevated serum creatinine, diabetes, or chronic inflammatory conditions. Subjects completed three research visits within nine months. Microalbuminuria was defined as the geometric mean ACR of 25–355 mg/g for women and 17–250 mg/g for men.
Results
The prevalence of microalbuminuria was 14%. The negative predictive value of a single urine ACR determination was 98%, whereas the positive predictive value was only 74%. Microalbuminuria was similar among Black (15%) and non-Black (14%) subjects (p=0.8). Subjects with microalbuminuria were more likely to have hypertension (p=0.02) and metabolic syndrome (p=0.03). While duration of HIV infection and the level of HIV viremia were similar between groups, those with microalbuminuria were more likely to have a CD4 count <200 cells/μL (p=0.0003). In a multivariate logistic regression analysis, the only significant independent predictors of microalbuminuria were low CD4 count (p=0.018) and current ritonavir exposure (p=0.04).
Conclusion
The prevalence of microalbuminuria in an HIV-infected clinic population was similar to earlier reports, and was associated with hypertension and impaired immune function. A single normal ACR determination effectively excludes microalbuminuria, whereas an elevated ACR requires confirmation.
doi:10.1159/000350384
PMCID: PMC3809894  PMID: 23615312
HIV infection; microalbuminuria; urinary albumin-creatinine ratio
8.  RECENT PROGRESS IN THE PATHOPHYSIOLOGY AND TREATMENT OF FSGS RECURRENCE 
Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized by proteinuria, frequent progression to end-stage renal disease, and recurrence after kidney transplantation in ~25% of patients, which negatively impacts long-term allograft survival. Experimental studies suggest that abnormalities in T and, possibly, B cells may represent one initial pathogenic trigger, leading to podocyte injury and progressive loss. New data also support the existence of circulating permeability factors able to damage the podocytes, but no single molecule has been consistently identified as the causal pathogenic element in FSGS recurrence. Unfortunately, major progress from mechanistic studies has not translated into substantial advancements in patient treatment, with plasmapheresis (PP) and high doses of cyclosporine (CsA) remaining the mainstays of therapy. Despite consistent experimental and clinical evidence that treatment of proteinuria slows renal function decline in proteinuric nephropathies, maximal use of antiproteinuric agents such as renin angiotensin system antagonists is not routine in the management of FSGS recurrence. More recently, encouraging results have been reported with anti-CD20 depleting antibody rituximab, but further studies are needed to establish its safety/efficacy profile.
doi:10.1111/ajt.12045
PMCID: PMC3558619  PMID: 23312002
kidney transplant; FSGS; glomerulonephritis; permeability factor; proteinuria
9.  TGF-beta1 reduces Wilms' tumor suppressor gene expression in podocytes 
Nephrology Dialysis Transplantation  2011;26(9):2746-2752.
Background. Wilms' tumor suppressor gene (WT1) is essential for normal podocyte function, and transforming growth factor (TGF)-beta contributes to focal segmental glomerulosclerosis (FSGS). We aimed to address whether TGF-beta affects WT1 expression in podocytes.
Methods. A human podocyte cell line treated with TGF-beta1 and kidneys in Alb/TGF-beta1-transgenic mice were analyzed for WT1 expression.
Results. In cultured podocytes, TGF-beta1 reduced WT1 protein expression determined by western blotting beginning at 8 h and decreased WT1 messenger RNA (mRNA) expression measured by quantitative reverse transcription–polymerase chain reaction beginning at 3 h. Knockdown of Smad4 by small hairpin (sh) RNA partially rescued the TGF-beta1-induced reduction of both WT1 protein and mRNA expressions in the cultured podocytes. TGF-beta1 did not alter luciferase activity of the reporter construct for a human WT1 promoter but reduced that for a human WT1 5′ enhancer construct, suggesting that TGF-beta1 may regulate WT1 expression by altering the 5′ enhancer activity. In the transgenic mice, WT1 protein expression in podocytes was decreased at 1 and 3 weeks of age, while glomeruloclerosis developed after 3 weeks.
Conclusion. TGF-beta1 reduces WT1 expression in cultured human podocytes and podocytes in mice before overt glomerulosclerosis begins. The effects are at least partially Smad4 dependent. Our findings identify a novel pathway linking TGF-beta1 to podocyte injury and FSGS. The WT1 reduction may be a useful marker for early podocyte injury.
doi:10.1093/ndt/gfr061
PMCID: PMC3175051  PMID: 21378152
FSGS; podocytes; TGF-beta1; Wilms' tumor suppressor gene
10.  Off the Beaten Renin–Angiotensin–Aldosterone System Pathway: New Perspectives on Antiproteinuric Therapy 
CKD is a major public health problem in the developed and the developing world. The degree of proteinuria associated with renal failure is a generally well accepted marker of disease severity. Agents with direct antiproteinuric effects are highly desirable therapeutic strategies for slowing, or even halting, progressive loss of kidney function. We review progress on therapies acting further downstream of the renin–angiotensin–aldosterone system pathway (e.g., transforming growth factor-beta antagonism, endothelin antagonism) and on those acting independent of the renin–angiotensin–aldosterone system pathway. In all, we discuss 26 therapeutic targets or compounds and 2 lifestyle changes (dietary modification and weight loss) that have been used clinically for diabetic or nondiabetic kidney disease. These therapies include endogenous molecules (estrogens, isotretinoin), biologic antagonists (monoclonal antibodies, soluble receptors), and small molecules. Where mechanistic data are available, these therapies have been shown to exert favorable effects on glomerular cell phenotype. In some cases, recent work has indicated surprising new molecular pathways for some therapies, such as direct effects on the podocyte by glucocorticoids, rituximab, and erythropoietin. It is hoped that recent advances in the basic science of kidney injury will prompt development of more effective pharmaceutical and biologic therapies for proteinuria.
doi:10.1053/j.ackd.2011.06.002
PMCID: PMC3245863  PMID: 21782136
Proteinuria; Albuminuria; Podocyte; Glomerulus; Diabetes; Novel therapies
11.  Apolipoprotein L1 gene variants associate with hypertension-attributed nephropathy and the rate of kidney function decline in African Americans 
Kidney international  2012;83(1):114-120.
Despite intensive anti-hypertensive therapy there was a high incidence of renal end-points in participants of the African American Study of Kidney Disease and Hypertension (AASK) cohort. To better understand this, coding variants in the apolipoprotein L1 (APOL1) and the non-muscle myosin heavy chain 9 (MYH9) genes were evaluated for an association with hypertension-attributed nephropathy and clinical outcomes in a case-control study. Clinical data and DNA were available for 675 AASK participant cases and 618 African American non-nephropathy control individuals. APOL1 G1 and G2, and MYH9 E1 variants along with 44 ancestry informative markers were genotyped with allele frequency differences between cases and controls analyzed by logistic regression multivariable models adjusting for ancestry, age, and gender. In recessive models, APOL1 risk variants were significantly associated with kidney disease in all cases compared to controls with an odds ratio of 2.57. In AASK cases with more advanced disease, such as a baseline urine protein to creatinine ratio over 0.6 g/g or a serum creatinine over 3 mg/dL during follow-up, the association was strengthened with odds ratios of 6.29 and 4.61, respectively. APOL1 risk variants were consistently associated with renal disease progression across medication classes and blood pressure targets. Thus, kidney disease in AASK participants was strongly associated with APOL1 renal risk variants.
doi:10.1038/ki.2012.263
PMCID: PMC3484228  PMID: 22832513
12.  HIV-associated nephropathy patients with and without apolipoprotein L1 gene variants have similar clinical and pathologic characteristics 
Kidney international  2012;82(3):338-343.
Recently, an association was found between non-diabetic kidney disease in African Americans and two independent sequence variants in the APOL1 gene, encoding apolipoprotein L1. In this study we determined the frequency of APOL1 risk variants in patients with biopsy-proven HIV-associated nephropathy (HIVAN) and distinctive pathological characteristics potentially driven by those risk variants. Among 76 patients with HIVAN, 60 were successfully genotyped for APOL1 G1 and G2 polymorphisms. In this cohort, 37 had two risk alleles, 18 were heterozygous and 5 had neither risk variant. There were no differences in the pathological findings of HIVAN and the number of APOL1 risk alleles. Further, the progression to end stage kidney disease or death did not differ by the number of risk alleles. Median renal survival was 9.3 months in patients with none or one risk allele compared to 11.7 months in patients with two APOL1 risk alleles. Thus, our study suggests that although the majority of African American patients with HIVAN have two APOL1 risk alleles, other as yet unknown factors in the host including genetic risk variants and environmental or viral factors may influence the development of this disorder in those with none or one APOL1 risk allele.
doi:10.1038/ki.2012.111
PMCID: PMC3463138  PMID: 22495294
13.  Crucial Roles of the Protein Kinases MK2 and MK3 in a Mouse Model of Glomerulonephritis 
PLoS ONE  2013;8(1):e54239.
Elevated mitogen-activated protein kinase p38 (p38 MAPK) signaling has been implicated in various experimental and human glomerulopathies, and its inhibition has proven beneficial in animal models of these diseases. p38 MAPK signaling is partially mediated through MK2 and MK3, two phylogenetically related protein kinases that are its direct substrates. The current study was designed to determine the specific roles of MK2 and MK3 in a mouse model of acute proliferative glomerulonephritis, using mice with disrupted MK2 and/or MK3 genes. We found that the absence of MK3 alone worsened the disease course and increased mortality slightly compared to wild-type mice, whereas the absence of MK2 alone exhibited no significant effect. However, in an MK3-free background, the disease course depended on the presence of MK2 in a gene dosage-dependent manner, with double knock-out mice being most susceptible to disease induction. Histological and renal functional analyses confirmed kidney damage following disease induction. Because the renal stress response plays a crucial role in kidney physiology and disease, we analyzed the stress response pattern in this disease model. We found that renal cortices of diseased mice exhibited a pronounced and specific pattern of expression and/or phosphorylation of stress proteins and other indicators of the stress response (HSPB1, HSPB6, HSPB8, CHOP, eIF2α), partially in a MK2/MK3 genotype-specific manner, and without induction of a general stress response. Similarly, the expression and activation patterns of other protein kinases downstream of p38 MAPK (MNK1, MSK1) depended partially on the MK2/MK3 genotype in this disease model. In conclusion, MK2 and MK3 together play crucial roles in the regulation of the renal stress response and in the development of glomerulonephritis, which can potentially be exploited to develop novel therapeutic approaches to treat glomerular disease.
doi:10.1371/journal.pone.0054239
PMCID: PMC3553169  PMID: 23372691
14.  Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes 
American journal of nephrology  2008;29(3):153-163.
Background
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
Methods
Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bearing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes.
Results
AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers.
Conclusion
Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
doi:10.1159/000151770
PMCID: PMC2698022  PMID: 18753740
Tetracycline-inducible system; Conditional immortalization; Transcription; Gene of interest
15.  Chronic kidney disease-induced HMGB1 elevation worsens sepsis and sepsis-induced acute kidney injury 
Kidney international  2011;80(11):1198-1211.
We previously showed that kidney dysfunction/interstitial fibrosis by folate predisposes mice to sepsis mortality (normal/sepsis: 15%; folate/sepsis: 90%); agents that increased survival in normal septic mice were ineffective in the two-stage model. We used a recently characterized 5/6 nephrectomy (Nx) mouse model of progressive chronic kidney disease (CKD) to study how CKD impacts sepsis and acute kidney injury (AKI) induced by cecal ligation-puncture (CLP). CKD intensified sepsis severity (by kidney and liver injury, cytokines, and spleen apoptosis). Accumulation of HMGB1, VEGF, TNF-α, IL-6, or IL-10 was increased in CKD or sepsis alone and to a greater extent in CKD-sepsis, and only part of this effect could be explained by decreased renal clearance. Surprisingly, we found splenic apoptosis in CKD, even in the absence of sepsis. Although sFLT-1 effectively treated sepsis, it was ineffective against CKD-sepsis. Conversely, a single dose of HMGB1-neutralizing antiserum, administered 6h after sepsis alone was ineffective; however, CKD/sepsis was attenuated by anti-HMGB1. Splenectomy transiently decreased circulating HMGB1 levels, which reversed the effectiveness of anti-HMGB1 treatment on CKD/sepsis. We conclude that progressive CKD increases sepsis severity, in part, by reducing renal clearance of cytokines; CKD-induced splenic apoptosis and HMGB1 could be important common mediators for both CKD and sepsis.
doi:10.1038/ki.2011.261
PMCID: PMC3491658  PMID: 21832986
17.  The Non-Muscle Myosin Heavy Chain 9 Gene (MYH9) Is Not Associated with Lupus Nephritis in African Americans 
American Journal of Nephrology  2010;32(1):66-72.
Background
African Americans (AA) disproportionately develop lupus nephritis (LN) relative to European Americans and familial clustering supports causative genes. Since MYH9 underlies approximately 40% of end-stage renal disease (ESRD) in AA, we tested for genetic association with LN.
Methods
Seven MYH9 single nucleotide polymorphisms (SNPs) and the E1 risk haplotype were tested for association with LN in three cohorts of AA.
Results
A preliminary analysis revealed that the MYH9 E1 risk haplotype was associated with ESRD in 25 cases with presumed systemic lupus erythematosus (SLE)-associated ESRD, compared to 735 non-SLE controls (odds ratio 3.1; p = 0.010 recessive). Replication analyses were performed in 583 AA with SLE in the PROFILE cohort (318 with LN; 265 with SLE but without nephropathy) and 60 AA from the NIH (39 with LN; 21 with SLE but without nephropathy). Analysis of the NIH and larger PROFILE cohorts, as well as a combined analysis, did not support this association.
Conclusions
These results suggest that AA with ESRD and coincident SLE who were recruited from dialysis clinics more likely have kidney diseases in the MYH9-associated spectrum of focal segmental glomerulosclerosis. PROFILE and NIH participants, recruited from rheumatology practices, demonstrate that MYH9 does not contribute substantially to the development of LN in AA.
doi:10.1159/000314688
PMCID: PMC2914393  PMID: 20523037
African Americans; Genetics; Lupus nephritis; Kidney; MYH9; Systemic lupus erythematosus
18.  Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes 
American Journal of Nephrology  2008;29(3):153-163.
Background
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
Methods
Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bearing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes.
Results
AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers.
Conclusion
Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
doi:10.1159/000151770
PMCID: PMC2698022  PMID: 18753740
Tetracycline-inducible system; Conditional immortalization; Transcription; Gene of interest
19.  Preventing renal and cardiovascular risk by renal function assessment: insights from a cross-sectional study in low-income countries and the USA 
BMJ Open  2012;2(5):e001357.
Objective
To assess the prevalence of microalbuminuria and kidney dysfunction in low-income countries and in the USA.
Design
Cross-sectional study of screening programmes in five countries.
Setting
Screening programmes in Nepal, Bolivia, the USA (National Health and Nutrition Examination Survey (NHANES) 2005–2008) Bangladesh and Georgia.
Participants
General population in Nepal (n=20 811), Bolivia (n=3436) and in the USA (n=4299) and high-risk subjects in Bangladesh (n=1518) and Georgia (n=1549).
Primary and secondary outcome measures
Estimated glomerular filtration rate (eGFR)<60ml/min/1.73 m2 and microalbuminuria (defined as urinary albumin creatinine ratio values of 30–300 mg/g) were the main outcome measures. The cardiovascular (CV) risk was also evaluated on the basis of demographic, clinical and blood data.
Results
The prevalence of eGFR<60ml/min/1.73 m2 was 19%, 3.2% and 7% in Nepal, Bolivia and the USA, respectively. In Nepal, 7% of subjects were microalbuminuric compared to 8.6% in the USA. The prevalence of participants with predicted 10-year CV disease (CVD) risk ≥10% was 16.9%, 9.4% and 17% in Nepal, Bolivia and in the USA, respectively. In Bangladesh and Georgia, subjects with eGFR<60 ml/min/1.73 m2 were 8.6% and 4.9%, whereas those with microalbuminuria were 45.4% and 56.5%, respectively. Predicted 10-year CVD risk ≥10% was 25.4% and 25% in Bangladesh and Georgia, respectively.
Conclusions
Renal abnormalities are common among low-income countries and in the USA. Prevention programmes, particularly focused on those with renal abnormalities, should be established worldwide to prevent CVD and progression to end-stage renal disease.
doi:10.1136/bmjopen-2012-001357
PMCID: PMC3467605  PMID: 23002161
20.  Longitudinal Assessment of Metabolic Abnormalities in Adolescents and Young Adults with HIV-infection Acquired Perinatally or in Early Childhood 
Objective
Metabolic complications of HIV pose challenges for health maintenance among young adults who acquired HIV in early childhood.
Materials/Methods
Between 7/2004–7/2009 we evaluated 47 HIV-infected subjects who acquired HIV in early life. Participants completed glucose tolerance testing, insulin, lipid, urine albumin and creatinine determinations and DXA scans. Longitudinal data were available for 39 subjects; duration of follow-up was 26.4±16.8 months.
Results
At baseline, participants were 17.1±3.9y and duration of antiretroviral therapy was 12.7±3.4y. CD4 count was 658±374 cells/mm3 and 55% had undetectable viral load. Impaired glucose tolerance (IGT) was present in 15%; 33% had insulin resistance (HOMA-IR>4.0). Further, 52% had triglycerides ≥150 mg/dL, 36% had HDL-c <40 mg/dl, 18% had LDL-c ≥130 mg/dL and 25% had total cholesterol ≥200 mg/dL. Microalbuminuria was present in 15% of participants and was inversely correlated with CD4% (p=0.001). During follow-up more than one third remained insulin resistant; lipid parameters tended to improve. There were significant increases in BMI (p=0.0002), percent leg fat (p=0.008) and trunk fat (p=0.002).
Conclusions
IGT, insulin resistance, dyslipidemia and microalbuminuria are common among young adults with HIV. Long-term exposure to therapy may translate into substantial persistent metabolic risk.
doi:10.1016/j.metabol.2010.08.007
PMCID: PMC3021796  PMID: 20947103
antiretroviral therapy; insulin resistance; dyslipidemia; microalbuminuria
21.  The Epidermal Growth Factor Receptor Promotes Glomerular Injury and Renal Failure in Rapidly Progressive Crescentic Glomerulonephritis; the Identification of Possible Therapy 
Nature Medicine  2011;17(10):1242-1250.
Rapidly progressive glomerulonephritis (RPGN) is a clinical a morphological expression of severe glomerular injury. Glomerular injury manifests as a proliferative histological pattern (“crescents”) with accumulation of T cells and macrophages, and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the EGFR/ErbB1 receptor in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 days after the induction of experimental RPGN. This suggests that targeting the HB-EGF/EGFR pathway could also be beneficial for treatment of human RPGN.
doi:10.1038/nm.2491
PMCID: PMC3198052  PMID: 21946538
22.  Trophoblast Glycoprotein: Possible Candidate Mediating Podocyte Injuries in Glomerulonephritis 
American Journal of Nephrology  2010;32(6):505-521.
Background
Trophoblast glycoprotein (Tpbg), a 72-kDa transmembrane glycoprotein, is known to regulate the phenotypes of epithelial cells by modifying actin organization and cell motility. Recently, a microarray study showed that Tpbg is upregulated in Thy1 glomerulonephritis (Thy1 GN). We hypothesized that Tpbg regulates cytoskeletal rearrangement and modulates phenotypic alteration in podocytes under pathological conditions.
Methods
We examined Tpbg expression in Thy1 GN and Tpbg function in mouse podocytes.
Results
We demonstrated that Tpbg is upregulated in the injured podocytes of Thy1 GN. In vitro, immunofluorescence studies revealed that Tpbg colocalized with the focal adhesion protein, vinculin, in parallel with stress fiber formation. This colocalization was observed even when actin filaments were depolymerized with cytochalasin D. Tpbg localization at focal adhesions was induced by dominant-active RhoA and suppressed by the ROCK1 inhibitor Y-26732. In addition, transforming growth factor-β increased Tpbg expression at focal adhesions concurrently with rearrangement of stress fibers. Stress fiber formation was suppressed in differentiated podocytes transfected with full-length Tpbg. Furthermore, knockdown of Tpbg using small interfering RNA decreased podocyte motility.
Conclusion
Our findings suggest a novel role of Tpbg in the phenotypic alteration of injured podocytes, and we accordingly propose a new mechanism of glomerular injury in glomerulonephritis.
doi:10.1159/000321366
PMCID: PMC2992649  PMID: 20980737
Trophoblast glycoprotein; Podocyte; Thy1 glomerulonephritis; Transforming growth factor-β; Cell motility; Stress fiber
23.  Solid-phase Synthesis and Screening of N-Acylated Polyamine (NAPA) Combinatorial Libraries for Protein Binding 
Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein’s interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV that is involved in numerous interactions with other proteins critical for viral replication.
doi:10.1016/j.bmcl.2010.09.054
PMCID: PMC2982675  PMID: 20932761
24.  Diabetic Nephropathy Is Accelerated by Farnesoid X Receptor Deficiency and Inhibited by Farnesoid X Receptor Activation in a Type 1 Diabetes Model 
Diabetes  2010;59(11):2916-2927.
OBJECTIVE
The pathogenesis of diabetic nephropathy is complex and involves activation of multiple pathways leading to kidney damage. An important role for altered lipid metabolism via sterol regulatory element binding proteins (SREBPs) has been recently recognized in diabetic kidney disease. Our previous studies have shown that the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor, modulates renal SREBP-1 expression. The purpose of the present study was then to determine if FXR deficiency accelerates type 1 diabetic nephropathy in part by further stimulation of SREBPs and related pathways, and conversely, if a selective FXR agonist can prevent the development of type 1 diabetic nephropathy.
RESEARCH DESIGN AND METHODS
Insulin deficiency and hyperglycemia were induced with streptozotocin (STZ) in C57BL/6 FXR KO mice. Progress of renal injury was compared with nephropathy-resistant wild-type C57BL/6 mice given STZ. DBA/2J mice with STZ-induced hyperglycemia were treated with the selective FXR agonist INT-747 for 12 weeks. To accelerate disease progression, all mice were placed on the Western diet after hyperglycemia development.
RESULTS
The present study demonstrates accelerated renal injury in diabetic FXR KO mice. In contrast, treatment with the FXR agonist INT-747 improves renal injury by decreasing proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis, and modulating renal lipid metabolism, macrophage infiltration, and renal expression of SREBPs, profibrotic growth factors, and oxidative stress enzymes in the diabetic DBA/2J strain.
CONCLUSIONS
Our findings indicate a critical role for FXR in the development of diabetic nephropathy and show that FXR activation prevents nephropathy in type 1 diabetes.
doi:10.2337/db10-0019
PMCID: PMC2963551  PMID: 20699418
25.  Increased Prevalence of Albuminuria in HIV-Infected Adults with Diabetes 
PLoS ONE  2011;6(9):e24610.
Objective
HIV and type 2 diabetes are known risk factors for albuminuria, but no previous reports have characterized albuminuria in HIV-infected patients with diabetes.
Research Design and Methods
We performed a cross-sectional study including 73 HIV-infected adults with type 2 diabetes, 82 HIV-infected non-diabetics, and 61 diabetic control subjects without HIV. Serum creatinine >1.5 mg/dL was exclusionary. Albuminuria was defined as urinary albumin/creatinine ratio >30 mg/g.
Results
The prevalence of albuminuria was significantly increased among HIV-infected diabetics (34% vs. 13% of HIV non-diabetic vs. 16% diabetic control, p = 0.005). HIV status and diabetes remained significant predictors of albuminuria after adjusting for age, race, BMI, and blood pressure. Albumin/creatinine ratio correlated significantly with HIV viral load (r = 0.28, p = 0.0005) and HIV-infected subjects with albuminuria had significantly greater cumulative exposure to abacavir (p = 0.01). In an adjusted multivariate regression analysis of HIV-infected subjects, the diagnosis of diabetes (p = 0.003), higher HIV viral load (p = 0.03) and cumulative exposure to abacavir (p = 0.0009) were significant independent predictors of albuminuria.
Conclusions
HIV and diabetes appear to have additive effects on albuminuria which is also independently associated with increased exposure to abacavir and HIV viral load. Future research on the persistence, progression and management of albuminuria in this unique at-risk population is needed.
doi:10.1371/journal.pone.0024610
PMCID: PMC3172239  PMID: 21931772

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