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1.  Mice with Hypomorphic Expression of the Sodium-Phosphate Cotransporter PiT1/Slc20a1 Have an Unexpected Normal Bone Mineralization 
PLoS ONE  2013;8(6):e65979.
The formation of hydroxyapatite crystals and their insertion into collagen fibrils of the matrix are essential steps for bone mineralization. As phosphate is a main structural component of apatite crystals, its uptake by skeletal cells is critical and must be controlled by specialized membrane proteins. In mammals, in vitro studies have suggested that the high-affinity sodium-phosphate cotransporter PiT1 could play this role. In vivo, PiT1 expression was detected in hypertrophic chondrocytes of murine metatarsals, but its implication in bone physiology is not yet deciphered. As the complete deletion of PiT1 results in embryonic lethality at E12.5, we took advantage of a mouse model bearing two copies of PiT1 hypomorphic alleles to study the effect of a low expression of PiT1 on bone mineralization in vivo. In this report, we show that a 85% down-regulation of PiT1 in long bones resulted in a slight (6%) but significant reduction of femur length in young mice (15- and 30-day-old). However, despite a defect in alcian blue / alizarin red S and Von Kossa staining of hypomorphic 1-day-old mice, using X-rays micro-computed tomography, energy dispersive X-ray spectroscopy and histological staining techniques we could not detect differences between hypomorphic and wild-type mice of 15- to 300-days old. Interestingly, the expression of PiT2, the paralog of PiT1, was increased 2-fold in bone of PiT1 hypomorphic mice accounting for a normal phosphate uptake in mutant cells. Whether this may contribute to the absence of bone mineralization defects remains to be further deciphered.
PMCID: PMC3681848  PMID: 23785462
2.  Determination of the best method to estimate glomerular filtration rate from serum creatinine in adult patients with sickle cell disease: a prospective observational cohort study 
BMC Nephrology  2012;13:83.
Sickle cell disease (SCD) leads to tissue hypoxia resulting in chronic organ dysfunction including SCD associated nephropathy. The goal of our study was to determine the best equation to estimate glomerular filtration rate (GFR) in SCD adult patients.
We conducted a prospective observational cohort study. Since 2007, all adult SCD patients in steady state, followed in two medical departments, have had their GFR measured using iohexol plasma clearance (gold standard). The Cockcroft-Gault, MDRD-v4, CKP-EPI and finally, MDRD and CKD-EPI equations without adjustment for ethnicity were tested to estimate GFR from serum creatinine. Estimated GFRs were compared to measured GFRs according to the graphical Bland and Altman method.
Sixty-four SCD patients (16 men, median age 27.5 years [range 18.0-67.5], 41 with SS-genotype were studied. They were Sub-Saharan Africa and French West Indies natives and predominantly lean (median body mass index: 22 kg/m2 [16-33]). Hyperfiltration (defined as measured GFR >110 mL/min/1.73 m2) was detected in 53.1% of patients. Urinary albumin/creatinine ratio was higher in patients with hyperfiltration than in patients with normal GFR (4.05 mg/mmol [0.14-60] versus 0.4 mg/mmol [0.7-81], p = 0.01). The CKD-EPI equation without adjustment for ethnicity had both the lowest bias and the greatest precision. Differences between estimated GFRs using the CKP-EPI equation and measured GFRs decreased with increasing GFR values, whereas it increased with the Cockcroft-Gault and MDRD-v4 equations.
We confirm that SCD patients have a high rate of glomerular hyperfiltration, which is frequently associated with microalbuminuria or macroalbuminuria. In non-Afro-American SCD patients, the best method for estimating GFR from serum creatinine is the CKD-EPI equation without adjustment for ethnicity. This equation is particularly accurate to estimate high GFR values, including glomerular hyperfiltration, and thus should be recommended to screen SCD adult patients at high risk for SCD nephropathy.
PMCID: PMC3465224  PMID: 22866669
Sickle cell disease; Glomerular hyperfiltration; Albuminuria; Glomerular filtration rate; CKD-EPI equation; Iohexol plasma clearance; Ethnicity
3.  A transcriptional network underlies susceptibility to kidney disease progression 
EMBO Molecular Medicine  2012;4(8):825-839.
The molecular networks that control the progression of chronic kidney diseases (CKD) are poorly defined. We have recently shown that the susceptibility to development of renal lesions after nephron reduction is controlled by a locus on mouse chromosome 6 and requires epidermal growth factor receptor (EGFR) activation. Here, we identified microphthalmia-associated transcription factor A (MITF-A), a bHLH-Zip transcription factor, as a modifier of CKD progression. Sequence analysis revealed a strain-specific mutation in the 5′ UTR that decreases MITF-A protein synthesis in lesion-prone friend virus B NIH (FVB/N) mice. More importantly, we dissected the molecular pathway by which MITF-A modulates CKD progression. MITF-A interacts with histone deacetylases to repress the transcription of TGF-α, a ligand of EGFR, and antagonizes transactivation by its related partner, transcription factor E3 (TFE3). Consistent with the key role of this network in CKD, Tgfa gene inactivation protected FVB/N mice from renal deterioration after nephron reduction. These data are relevant to human CKD, as we found that the TFE3/MITF-A ratio was increased in patients with damaged kidneys. Our study uncovers a novel transcriptional network and unveils novel potential prognostic and therapeutic targets for preventing human CKD progression.
PMCID: PMC3494079  PMID: 22711280
EGFR; genetic susceptibility; MITF-A; renal lesions; TGF-alpha
4.  Functional Interaction between CFTR and the Sodium-Phosphate Co-Transport Type 2a in Xenopus laevis Oocytes 
PLoS ONE  2012;7(4):e34879.
A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.
NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.
We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis.
PMCID: PMC3325942  PMID: 22514683
5.  A New Human NHERF1 Mutation Decreases Renal Phosphate Transporter NPT2a Expression by a PTH-Independent Mechanism 
PLoS ONE  2012;7(4):e34764.
The sodium-hydrogen exchanger regulatory factor 1 (NHERF1) binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH) receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule.
We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production.
We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A) located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a.
Our results indicate that the PDZ1 domain is critical for NHERF1- NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.
PMCID: PMC3323571  PMID: 22506049
6.  Lipocalin 2 is essential for chronic kidney disease progression in mice and humans 
The Journal of Clinical Investigation  2010;120(11):4065-4076.
Mechanisms of progression of chronic kidney disease (CKD), a major health care burden, are poorly understood. EGFR stimulates CKD progression, but the molecular networks that mediate its biological effects remain unknown. We recently showed that the severity of renal lesions after nephron reduction varied substantially among mouse strains and required activation of EGFR. Here, we utilized two mouse strains that react differently to nephron reduction — FVB/N mice, which develop severe renal lesions, and B6D2F1 mice, which are resistant to early deterioration — coupled with genome-wide expression to elucidate the molecular nature of CKD progression. Our results showed that lipocalin 2 (Lcn2, also known as neutrophil gelatinase–associated lipocalin [NGAL]), the most highly upregulated gene in the FVB/N strain, was not simply a marker of renal lesions, but an active player in disease progression. In fact, the severity of renal lesions was dramatically reduced in Lcn2–/– mice. We discovered that Lcn2 expression increased upon EGFR activation and that Lcn2 mediated its mitogenic effect during renal deterioration. EGFR inhibition prevented Lcn2 upregulation and lesion development in mice expressing a dominant negative EGFR isoform, and hypoxia-inducible factor 1α (Hif-1α) was crucially required for EGFR-induced Lcn2 overexpression. Consistent with this, cell proliferation was dramatically reduced in Lcn2–/– mice. These data are relevant to human CKD, as we found that LCN2 was increased particularly in patients who rapidly progressed to end-stage renal failure. Together our results uncover what we believe to be a novel function for Lcn2 and a critical pathway leading to progressive renal failure and cystogenesis.
PMCID: PMC2964970  PMID: 20921623
7.  The Phosphate Transporter PiT1 (Slc20a1) Revealed As a New Essential Gene for Mouse Liver Development 
PLoS ONE  2010;5(2):e9148.
PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na+-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.
Methodology/Principal Findings
To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5–E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.
This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.
PMCID: PMC2818845  PMID: 20161774
8.  JunD protects against chronic kidney disease by regulating paracrine mitogens 
Journal of Clinical Investigation  2003;112(6):843-852.
The AP-1 transcription factor, composed of Jun and Fos proteins, plays a crucial role in the fine tuning of cell proliferation. We showed previously that AP-1 complexes are activated during the proliferative response that parallels the development of renal lesions after nephron reduction, but little is known about the specific role of individual Jun/Fos components in the deterioration process. Here we used JunD knockout (JunD–/–) mice and an experimental model of chronic renal injury (75% nephron reduction) to explore the role of JunD. Nephron reduction resulted in an initial compensatory growth phase that did not require JunD. JunD, however, was essential to inhibit a second wave of cell proliferation and to halt the development of severe glomerular sclerosis, tubular dilation, and interstitial fibrosis. We show that the effects of junD inactivation are not cell autonomous and involve upregulation of the paracrine mitogen, TGF-α. Expression of a transgene (REM) encoding a dominant negative isoform of the EGFR, the receptor for TGF-α, prevented the second wave of cell proliferation and the development of renal lesions in bitransgenic JunD–/–/REM mice. We propose that JunD is part of a regulatory network that controls proliferation to prevent pathological progression in chronic renal diseases.
PMCID: PMC193664  PMID: 12975469
9.  Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstitial lesions after renal injury 
Journal of Clinical Investigation  2000;106(2):225-234.
The role of EGF in the evolution of renal lesions after injury is still controversial. To determine whether the EGF expression is beneficial or detrimental, we generated transgenic mice expressing a COOH-terminal–truncated EGF-R under the control of the kidney-specific type 1 γ-glutamyl transpeptidase promoter. As expected, the transgene was expressed exclusively at the basolateral membrane of proximal tubular cells. Under basal conditions, transgenic mice showed normal renal morphology and function. Infusion of EGF to transgenic animals revealed that the mutant receptor behaved in a dominant-negative manner and prevented EGF-signaled EGF-R autophosphorylation. We next evaluated the impact of transgene expression on the development of renal lesions in two models of renal injury. After 75% reduction of renal mass, tubular dilations were less severe in transgenic mice than in wild-type animals. After prolonged renal ischemia, tubular atrophy and interstitial fibrosis were reduced in transgenic mice as compared with wild-type mice. The beneficial effect of the transgene included a reduction of tubular cell proliferation, interstitial collagen accumulation, and mononuclear cell infiltration. In conclusion, functional inactivation of the EGF-R in renal proximal tubular cells reduced tubulo-interstitial lesions after renal injury. These data suggest that blocking the EGF pathway may be a therapeutic strategy to reduce the progression of chronic renal failure.
PMCID: PMC314303  PMID: 10903338

Results 1-9 (9)