Fabiola Terzi

Angiotensin II and EGF receptor cross-talk in chronic kidney diseases: a new therapeutic approach

Mechanisms of progression of chronic renal diseases, a major healthcare burden, are poorly understood. Angiotensin II (AngII), the major renin-angiotensin system effector, is known to be involved in renal deterioration, but the molecular pathways are still unknown. Here, we show that mice overexpressing a dominant negative isoform of epidermal growth factor receptor (EGFR) were protected from renal lesions during chronic AngII infusion. Transforming growth factor-α (TGF-α) and its sheddase, TACE (also known as ADAM17), were induced by AngII treatment, TACE was redistributed to apical membranes and EGFR was phosphorylated. AngII-induced lesions were substantially reduced in mice lacking TGF-α or in mice given a specific TACE inhibitor. Pharmacologic inhibition of AngII prevented TGF-α and TACE accumulation as well as renal lesions after nephron reduction. These findings indicate a crucial role for AngII-dependent EGFR transactivation in renal deterioration and identify in TACE inhibitors a new therapeutic strategy for preventing progression of chronic renal diseases.

Lipocalin 2 is essential for chronic kidney disease progression in mice and humans

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.

Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstitial lesions after renal injury

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 gamma-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.

Impaired flow-induced dilation in mesenteric resistance arteries from mice lacking vimentin.

The intermediate filament vimentin might play a key role in vascular resistance to mechanical stress. We investigated the responses to pressure (tensile stress) and flow (shear stress) of mesenteric resistance arteries perfused in vitro from vimentin knockout mice. Arteries were isolated from homozygous (Vim-/-, n = 14) or heterozygous vimentin-null mice (Vim+/-, n = 5) and from wild-type littermates (Vim+/+, n = 9). Passive arterial diameter (175+/-15 micron in Vim+/+ at 100 mmHg) and myogenic tone were not affected by the absence of vimentin. Flow-induced (0-150 microl/min) dilation (e. g., 19+/-3 micron dilation at 150 mmHg in Vim+/+) was significantly attenuated in Vim-/- mice (13+/-2 micron dilation, P < 0.01). Acute blockade of nitric oxide synthesis (NG-nitro- L-arginine, 10 microM) significantly decreased flow-induced dilation in both groups, whereas acute blockade of prostaglandin synthesis (indomethacin, 10 microM) had no significant effect. Mean blood pressure, in vivo mesenteric blood flow and diameter, and mesenteric artery media thickness or media to lumen ratio were not affected by the absence of vimentin. Thus, the absence of vimentin decreased selectively the response of resistance arteries to flow, suggesting a role for vimentin in the mechanotransduction of shear stress.

AKT2 is essential to maintain podocyte viability and function during chronic kidney disease

In chronic kidney disease (CKD), loss of functional nephrons results in metabolic and mechanical stress in the remaining ones, resulting in further nephron loss. Here we show that Akt2 activation has an essential role in podocyte protection after nephron reduction. Glomerulosclerosis and albuminuria were substantially worsened in Akt2−/− but not in Akt1−/− mice as compared to wild-type mice. Specific deletion of Akt2 or its regulator Rictor in podocytes revealed that Akt2 has an intrinsic function in podocytes. Mechanistically, Akt2 triggers a compensatory program that involves mouse double minute 2 homolog (Mdm2), glycogen synthase kinase 3 (Gsk3) and Rac1. The defective activation of this pathway after nephron reduction leads to apoptosis and foot process effacement of the podocytes. We further show that AKT2 activation by mammalian target of rapamycin complex 2 (mTORC2) is also required for podocyte survival in human CKD. More notably, we elucidate the events underlying the adverse renal effect of sirolimus and provide a criterion for the rational use of this drug. Thus, our results disclose a new function of Akt2 and identify a potential therapeutic target for preserving glomerular function in CKD.

A mitotic transcriptional switch in polycystic kidney disease

Hepatocyte nuclear factor-1β (HNF-1β) is a transcription factor required for the expression of several renal cystic genes and whose prenatal deletion leads to polycystic kidney disease (PKD). We show here that inactivation of Hnf1b from postnatal day 10 onward does not elicit cystic dilations in tubules after their proliferative morphogenetic elongation is over. Cystogenic resistance is intrinsically linked to the quiescent state of cells. In fact, when Hnf1b deficient quiescent cells are forced to proliferate by an ischemia-reperfusion injury, they give rise to cysts, owing to loss of oriented cell division. Remarkably, in quiescent cells, the transcription of crucial cystogenic target genes is maintained even in the absence of HNF-1β. However, their expression is lost as soon as cells proliferate and the chromatin of target genes acquires heterochromatin marks. These results unveil a previously undescribed aspect of gene regulation. It is well established that transcription is shut off during the mitotic condensation of chromatin. We propose that transcription factors such as HNF-1β might be involved in reprogramming gene expression after transcriptional silencing is induced by mitotic chromatin condensation. Notably, HNF-1β remains associated with the mitotically condensed chromosomal barrels. This association suggests that HNF-1β is a bookmarking factor that is necessary for reopening the chromatin of target genes after mitotic silencing.

Reduction of renal mass is lethal in mice lacking vimentin. Role of endothelin-nitric oxide imbalance.

Modulation of vascular tone by chemical and mechanical stimuli is a crucial adaptive phenomenon which involves cytoskeleton elements. Disruption, by homologous recombination, of the gene encoding vimentin, a class III intermediate filament protein mainly expressed in vascular cells, was reported to result in apparently normal phenotype under physiological conditions. In this study, we evaluated whether the lack of vimentin affects vascular adaptation to pathological situations, such as reduction of renal mass, a pathological condition which usually results in immediate and sustained vasodilation of the renal vascular bed. Ablation of 3/4 of renal mass was constantly lethal within 72 h in mice lacking vimentin (Vim-/-), whereas no lethality was observed in wild-type littermates. Death in Vim-/- mice resulted from end-stage renal failure. Kidneys from Vim-/- mice synthesized more endothelin, but less nitric oxide (NO), than kidneys from normal animals. In vitro, renal resistance arteries from Vim-/- mice were selectively more sensitive to endothelin, less responsive to NO-dependent vasodilators, and exhibited an impaired flow (shear stress)- induced vasodilation, which is NO dependent, as compared with those from normal littermates. Finally, in vivo administration of bosentan, an endothelin receptor antagonist, totally prevented lethality in Vim-/- mice. These results suggest that vimentin plays a key role in the modulation of vascular tone, possibly via the tuning of endothelin-nitric oxide balance.

Proliferation and Remodeling of the Peritubular Microcirculation after Nephron Reduction : Association with the Progression of Renal Lesions

Little is known about the serial changes that might occur in renal capillaries after reduction of renal mass. In the current study, our aim was to document potential alterations in the morphology and proliferation of the renal cortical peritubular microcirculation at specific time points (7 and 60 days) after experimental 75% surgical nephron reduction using two strains of mice that we here demonstrate react differently to the same initial insult: one strain (C57BL6xDBA2/F1 mice) undergoes compensatory growth alone, whereas the other (FVB/N mice) additionally develops severe tubulo-interstitial lesions. Our data demonstrate that significant remodeling and proliferation occur in renal cortical peritubular capillaries after experimental nephron reduction, as assessed by microangiography using infusion of fluorescein isothiocyanate-labeled dextran, expression of the endothelial markers CD34 and Tie-2, and co-expression of CD34 and proliferating cell nuclear antigen, a surrogate marker of cell proliferation. This was accompanied by an increase of renal vascular endothelial growth factor protein levels and a change in distribution of this protein within the kidney itself. Moreover, most of these responses were accentuated in FVB/N mice in the presence of progressive renal disease and positively correlated with tubular epithelial cell proliferation. Hence, we have made three significant novel observations that illuminate the complex pathophysiology of chronic kidney damage after nephron reduction: 1) cortical peritubular capillaries grow by proliferation and remodeling, 2) vascular endothelial growth factor expression is altered, and 3) the development of tubulo-interstitial disease is genetically determined.

Regulation of YAP by mTOR and autophagy reveals a therapeutic target of tuberous sclerosis complex

Liang et al. find that the tumor suppressors TSC1 and TSC2, defects in which underlie the genetic disease Tuberous Sclerosis Complex (TSC), drive the mTOR-dependent autophagosomal destruction of the transcriptional activator YAP. Blocking YAP inhibited the abnormal proliferation of TSC1/2-deficient human cells and reversed TSC-like disease symptoms in mosaic Tsc1 mutant mice.

ZONAB Promotes Proliferation and Represses Differentiation of Proximal Tubule Epithelial Cells

Epithelial polarization modulates gene expression. The transcription factor zonula occludens 1 (ZO-1)-associated nucleic acid binding protein (ZONAB) can shuttle between tight junctions and nuclei, promoting cell proliferation and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), but whether it also represses epithelial differentiation is unknown. Here, during mouse kidney ontogeny and polarization of proximal tubular cells (OK cells), ZONAB and PCNA levels decreased in parallel and inversely correlated with increasing apical differentiation, reflected by expression of megalin/cubilin, maturation of the brush border, and extension of the primary cilium. Conversely, ZONAB reexpression and loss of apical differentiation markers provided a signature for renal clear cell carcinoma. In confluent OK cells, ZONAB overexpression increased proliferation and PCNA while repressing megalin/cubilin expression and impairing differentiation of the brush border and primary cilium. Reporter and chromatin immunoprecipitation assays demonstrated that megalin and cubilin are ZONAB target genes. Sparsely plated OK cells formed small islands composed of distinct populations: Cells on the periphery, which lacked external tight junctions, strongly expressed nuclear ZONAB, proliferated, and failed to differentiate; central cells, surrounded by continuous junctions, lost nuclear ZONAB, stopped proliferating, and engaged in apical differentiation. Taken together, these data suggest that ZONAB is an important component of the mechanisms that sense epithelial density and participates in the complex transcriptional networks that regulate the switch between proliferation and differentiation.