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1.  Rethinking hypertensive kidney disease: arterionephrosclerosis as a genetic, metabolic, and inflammatory disorder 
Purpose of review
Hypertension is the attributed cause of approximately 30% of end-stage kidney disease cases in the United States, but there has been controversy as to whether benign hypertension is a cause of chronic kidney disease.
Recent findings
The histology of chronic kidney disease attributed to nonmalignant hypertension is arterionephrosclerosis, with pathology in the terminal branches of the interlobular arteries, together with global glomerulosclerosis. The identification of coding region variants in APOL1, encoding apolipoprotein L1, has opened a new perspective on this debate. These variants are restricted to populations of recent African descent and are strongly associated with clinically diagnosed arterionephrosclerosis, particularly when there is moderate-grade or high-grade proteinuria or progression to more advanced levels of kidney dysfunction. Nevertheless, not all African Americans with hypertension who progress to end-stage kidney disease have two APOL1 risk variants, and individuals of European and Asian descent also manifest arterionephrosclerosis. Further, we do not understand the mechanisms by which APOL1 initiates pathology in the renal microcirculation.
APOL1 nephropathy comprises a disease spectrum (perhaps with distinct endophenotypes), including focal segmental glomerulosclerosis, collapsing glomerulopathy, and arterionephrosclerosis. The terms hypertensive kidney disease and hypertensive nephrosclerosis have outlived their usefulness. It may be time to use the established, etiologically neutral term, arterionephrosclerosis, to consider whether this is a disease rather than a pathologic description, and to determine the causal role of various clinical correlates including aging, obesity, hyperlipidemia, smoking, chronic inflammation, and oxidative stress.
PMCID: PMC4165431  PMID: 23470819
apolipoprotein L1; arterionephrosclerosis; hypertensive kidney disease
2.  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.
PMCID: PMC4055698  PMID: 24924335
3.  PPARα and Sirt1 Mediate Erythropoietin Action in Increasing Metabolic Activity and Browning of White Adipocytes to Protect Against Obesity and Metabolic Disorders 
Diabetes  2013;62(12):4122-4131.
Erythropoietin (EPO) has shown beneficial effects in the regulation of obesity and metabolic syndrome; however, the detailed mechanism is still largely unknown. Here, we created mice with adipocyte-specific deletion of EPO receptor. These mice exhibited obesity and decreased glucose tolerance and insulin sensitivity, especially when fed a high-fat diet. Moreover, EPO increased oxidative metabolism, fatty acid oxidation, and key metabolic genes in adipocytes and in white adipose tissue from diet-induced obese wild-type mice. Increased metabolic activity by EPO is associated with induction of brown fat–like features in white adipocytes, as demonstrated by increases in brown fat gene expression, mitochondrial content, and uncoupled respiration. Peroxisome proliferator–activated receptor (PPAR)α was found to mediate EPO activity because a PPARα antagonist impaired EPO-mediated induction of brown fat–like gene expression and uncoupled respiration. PPARα also cooperates with Sirt1 activated by EPO through modulating the NAD+ level to regulate metabolic activity. PPARα targets, including PPARγ coactivator 1α, uncoupling protein 1, and carnitine palmitoyltransferase 1α, were increased by EPO but impaired by Sirt1 knockdown. Sirt1 knockdown also attenuated adipose response to EPO. Collectively, EPO, as a novel regulator of adipose energy homeostasis via these metabolism coregulators, provides a potential therapeutic strategy to protect against obesity and metabolic disorders.
PMCID: PMC3837041  PMID: 23990359
4.  Renal Growth in Isolated Methylmalonic Acidemia (MMA) 
We sought to predict renal growth based on clinical and metabolic parameters in patients with isolated methylmalonic acidemia (MMA), a group of disorders associated with chronic kidney disease.
Fifty MMA patients, followed from 2004 to 2011, were classified by molecular genetics and studied using a combined cross-sectional and longitudinal design that included renal ultrasound examinations, anthropometric measurements, and metabolic phenotyping. Renal length was compared to healthy controls and modeled to other clinical parameters using multiple regression analyses.
Comparisons with age-matched controls showed that renal length in MMA subjects was significantly decreased (p < 0.05). Stepwise regression modeling found that combinations of height, serum cystatin C, and serum methymalonic acid concentrations best predicted kidney size. The regression equations used to generate MMA kidney nomograms were: renal length (cm) = 6.79 + 0.22 * age for the controls and 6.80 + 0.09 * age for the MMA cohort (p < 0.001; constant and slope).
Renal length, reflective of kidney growth, significantly decreased in MMA patients over time compared to controls and was predictable with select clinical parameters. Cystatin C and serum methylmalonic acid concentrations were highly correlated with smaller kidneys and decreased renal function in this patient population.
PMCID: PMC4149057  PMID: 23639900
isolated methylmalonic acidemia; renal growth; renal ultrasound; chronic kidney disease; cystatin C; methylmalonyl-CoA mutase; vitamin B12
5.  Viruses and collapsing glomerulopathy: a brief critical review 
Clinical Kidney Journal  2012;6(1):1-5.
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.
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).
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.
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.
PMCID: PMC3560379  PMID: 23372939
collapsing focal segmental glomerulosclerosis; cytomegalovirus; Epstein-Barr virus; parvovirus B19; podocyte
6.  Integrated Design of Antibodies for Systems Biology Using Ab Designer 
In the current era of large-scale biology, systems biology has evolved as a powerful approach to identify complex interactions within biological systems. In addition to high throughput identification and quantification techniques, methods based on high-quality mono-specific antibodies remain an essential element of the approach. To assist the large-scale design and production of peptide-directed antibodies for systems biology studies, we developed a fully integrated online application, AbDesigner (, to help researchers select optimal peptide immunogens for antibody generation against relatively disordered regions of target proteins. Here we describe AbDesigner in terms of its features, comparing it to other software tools, and use it to design three antibodies against kidney disease-related proteins in human, viz. nephrin, podocin, and apolipoprotein L1.
PMCID: PMC4201049  PMID: 25328345
Antibody design; Software tools; Systems biology
7.  Podocyte Injury Caused by Indoxyl Sulfate, a Uremic Toxin and Aryl-Hydrocarbon Receptor Ligand 
PLoS ONE  2014;9(9):e108448.
Indoxyl sulfate is a uremic toxin and a ligand of the aryl-hydrocarbon receptor (AhR), a transcriptional regulator. Elevated serum indoxyl sulfate levels may contribute to progressive kidney disease and associated vascular disease. We asked whether indoxyl sulfate injures podocytes in vivo and in vitro. Mice exposed to indoxyl sulfate for 8 w exhibited prominent tubulointerstitial lesions with vascular damage. Indoxyl sulfate-exposed mice with microalbuminuria showed ischemic changes, while more severely affected mice showed increased mesangial matrix, segmental solidification, and mesangiolysis. In normal mouse kidneys, AhR was predominantly localized to the podocyte nuclei. In mice exposed to indoxyl sulfate for 2 h, isolated glomeruli manifested increased Cyp1a1 expression, indicating AhR activation. After 8 w of indoxyl sulfate, podocytes showed foot process effacement, cytoplasmic vacuoles, and a focal granular and wrinkled pattern of podocin and synaptopodin expression. Furthermore, vimentin and AhR expression in the glomerulus was increased in the indoxyl sulfate-exposed glomeruli compared to controls. Glomerular expression of characteristic podocyte mRNAs was decreased, including Actn4, Cd2ap, Myh9, Nphs1, Nphs2, Podxl, Synpo, and Wt1. In vitro, immortalized-mouse podocytes exhibited AhR nuclear translocation beginning 30 min after 1 mM indoxyl sulfate exposure, and there was increased phospho-Rac1/Cdc42 at 2 h. After exposure to indoxyl sulfate for 24 h, mouse podocytes exhibited a pro-inflammatory phenotype, perturbed actin cytoskeleton, decreased expression of podocyte-specific genes, and decreased cell viability. In immortalized human podocytes, indoxyl sulfate treatment caused cell injury, decreased mRNA expression of podocyte-specific proteins, as well as integrins, collagens, cytoskeletal proteins, and bone morphogenetic proteins, and increased cytokine and chemokine expression. We propose that basal levels of AhR activity regulate podocyte function under normal conditions, and that increased activation of podocyte AhR by indoxyl sulfate contributes to progressive glomerular injury.
PMCID: PMC4171541  PMID: 25244654
8.  Systemic Diagnostic Testing in Patients With Apparently Isolated Uveal Coloboma 
American journal of ophthalmology  2013;156(6):1159-1168.e4.
To investigate the frequency and types of systemic findings in patients with apparently isolated uveal coloboma.
Cross-sectional observational study.
Single-center ophthalmic genetics clinic.
Ninety-nine patients with uveal coloboma seen at the National Eye Institute.
Results of audiology testing, echocardiogram, brain magnetic resonance imaging, renal ultrasound, and total spine radiographs.
Prevalence of abnormal findings on systemic testing.
Uveal coloboma affected only the anterior segment in 8 patients, only the posterior segment in 23 patients, and both anterior and posterior segments in 68 patients. Best-corrected visual acuity (BCVA) of eyes with coloboma was ≥20/40 in 45% of eyes; 23% of eyes had BCVA of ≤20/400. The majority of patients (74%) had good vision (>20/60) in at least 1 eye. Ten of the 19 patients (53%) who underwent echocardiography had abnormalities, with ventral septal defects being the most prevalent. Abnormal findings were observed in 5 of 72 patients (7%) who had a renal ultrasound and in 5 of 29 patients (17%) who underwent a brain MRI. Audiology testing revealed abnormalities in 13 of 75 patients (17%), and spine radiographs showed anomalies in 10 of 77 patients (13%). Most findings required no acute intervention.
Although some patients with coloboma had evidence of extraocular abnormalities, the majority of findings on routine clinical examination did not require acute intervention, but some warranted follow-up. Results from the systemic evaluation of patients with coloboma should be interpreted with caution and in view of their clinical context.
PMCID: PMC4167417  PMID: 24012100
9.  Increased mitochondrial activity in renal proximal tubule cells from young spontaneously hypertensive rats 
Kidney international  2013;85(3):561-569.
Renal proximal tubule cells from spontaneously hypertensive rats (SHR), compared with normotensive Wistar-Kyoto rats (WKY), have increased oxidative stress. The contribution of mitochondrial oxidative phosphorylation to the subsequent hypertensive phenotype remains unclear. We found that renal proximal tubule cells from SHR, relative to WKY, had significantly higher basal oxygen consumption rates, ATP synthesis-linked oxygen consumption rates, and maximum and reserve respiration. These bioenergetic parameters indicated increased mitochondrial function in renal proximal tubule cells from SHR compared with WKY. Pyruvate dehydrogenase complex activity was consistently higher in both renal proximal tubule cells and cortical homogenates from SHR than WKY. Treatment for 6 days with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase, significantly increased renal pyruvate dehydrogenase complex activity and systolic blood pressure in 3-week old WKY and SHR. Therefore, mitochondrial oxidative phosphorylation is higher in renal proximal tubule cells from SHR compared with WKY. Thus the pyruvate dehydrogenase complex is a determinant of increased mitochondrial metabolism that could be a causal contributor to the hypertension in SHR.
PMCID: PMC3943540  PMID: 24132210
oxygen consumption rate; pyruvate dehydrogenase complex; mitochondria; systolic blood pressure; renal proximal tubule
10.  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.
PMCID: PMC4096868  PMID: 17942957
11.  APOL1 Risk Variants, Race, and Progression of Chronic Kidney Disease 
The New England journal of medicine  2013;369(23):2183-2196.
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.
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.
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).
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.)
PMCID: PMC3969022  PMID: 24206458
12.  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.
PMCID: PMC3175051  PMID: 21378152
FSGS; podocytes; TGF-beta1; Wilms' tumor suppressor gene
13.  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.
PMCID: PMC4009012  PMID: 24285483
14.  Tenofovir Treatment Duration Predicts Proteinuria in a Multi-Ethnic United States Cohort of Children and Adolescents with Perinatal HIV-1 Infection 
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.
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.
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.
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.
PMCID: PMC3800277  PMID: 23249917
Tenofovir; proteinuria; chronic kidney disease; proximal tubules; nephrotoxicity; urine protein/creatinine ratio
15.  Microalbuminuria in HIV Disease 
American journal of nephrology  2013;37(5):10.1159/000350384.
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.
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.
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).
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.
PMCID: PMC3809894  PMID: 23615312
HIV infection; microalbuminuria; urinary albumin-creatinine ratio
16.  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.
PMCID: PMC3245863  PMID: 21782136
Proteinuria; Albuminuria; Podocyte; Glomerulus; Diabetes; Novel therapies
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.
PMCID: PMC3558619  PMID: 23312002
kidney transplant; FSGS; glomerulonephritis; permeability factor; proteinuria
18.  Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes 
American journal of nephrology  2008;29(3):153-163.
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
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.
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.
Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
PMCID: PMC2698022  PMID: 18753740
Tetracycline-inducible system; Conditional immortalization; Transcription; Gene of interest
19.  Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes 
American Journal of Nephrology  2008;29(3):153-163.
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
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.
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.
Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
PMCID: PMC2698022  PMID: 18753740
Tetracycline-inducible system; Conditional immortalization; Transcription; Gene of interest
20.  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.
PMCID: PMC3484228  PMID: 22832513
21.  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.
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.
Seven MYH9 single nucleotide polymorphisms (SNPs) and the E1 risk haplotype were tested for association with LN in three cohorts of AA.
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.
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.
PMCID: PMC2914393  PMID: 20523037
African Americans; Genetics; Lupus nephritis; Kidney; MYH9; Systemic lupus erythematosus
22.  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.
PMCID: PMC3463138  PMID: 22495294
23.  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.
PMCID: PMC3553169  PMID: 23372691
24.  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.
PMCID: PMC3491658  PMID: 21832986

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