Federica Mezzabotta

The renal stem cell system in kidney repair and regeneration.

The adult mammalian renal tubular epithelium exists in a relatively quiescent to slowly replicating state, but has great potential for regenerative morphogenesis following severe ischemic or toxic injury. Kidney regeneration and repair occur through three cellular and molecular mechanisms: differentiation of the somatic stem cells, recruitment of circulating stem cells and, more importantly, proliferation/dedifferentiation of mature cells. Dedifferentiation seems to represent a critical step for the recovery of tubular integrity. Dedifferentiation of tubular cells after injury is characterized by the reactivation of a mesenchymal program that is active during nephrogenesis. Epithelial-to-mesenchymal transition (EMT) of renal tubular cells is an extreme manifestation of epithelial cell plasticity. It is now widely recognized as a fundamental process that marks some physiological, such as morphogenesis, as well as pathological events, such as oncogenesis and fibrogenesis. It might be also considered as a key event in the regenerative process of the kidney. Understanding the molecular mechanisms involved in EMT might be useful for designing therapeutic strategies in order to potentiate the innate capacity of the kidney to regenerate.

The Regenerative Potential of the Kidney: What Can We Learn from Developmental Biology?

Cell turnover in the healthy adult kidney is very slow but the kidney has a strong capacity for regeneration after acute injury. Although many molecular aspects of this process have been clarified, the source of the newly-formed renal epithelial cells is still being debated. Several studies have shown, moreover, that the repair of injured renal epithelium starts from mature tubular cells, which enter into an activated proliferative state characterized by the reappearance of mesenchymal markers detectable during nephrogenesis, thus pointing to a marked plasticity of renal epithelial cells. The regenerative potential of mature epithelial cells might stem from their almost unique morphogenetic process. Unlike other tubular organs, all epithelial and mesenchymal cells in the kidney derive from the same germ layer, the mesoderm. In a fascinating view of vertebrate embryogenesis, the mesoderm might be seen as a cell layer capable of oscillating between epithelial and mesenchymal states, thus acquiring a remarkable plasticity that lends it an extended potential for innovation and a better control of three-dimensional body organization. The renal papilla contains a population of cells with the characteristic of adult stem cells. Mesenchymal stromal stem cells (MSC) have been found to reside in the connective tissue of most organs, including the kidney. Recent studies indicate that the MSC compartment extends throughout the body postnatally as a result of its perivascular location. Developmental biology suggests that this might be particularly true of the kidney and that the papilla might represent the perivascular renal stem cell niche. The perivascular niche hypothesis fits well with the evolving concept of the stem cell niche as an entity of action. It is its dynamic capability that makes the niche concept so important and essential to the feasibility of regenerative medicine.

Spontaneous calcification process in primary renal cells from a medullary sponge kidney patient harbouring a GDNF mutation

Medullary nephrocalcinosis is a hallmark of medullary sponge kidney (MSK). We had the opportunity to study a spontaneous calcification process in vitro by utilizing the renal cells of a patient with MSK who was heterozygous for the c.‐27 + 18G>A variant in the GDNF gene encoding glial cell‐derived neurotrophic factor. The cells were obtained by collagenase digestion of papillary tissues from the MSK patient and from two patients who had no MSK or nephrocalcinosis. These cells were typed by immunocytochemistry, and the presence of mineral deposits was studied using von Kossa staining, scanning electron microscopy analysis and an ALP assay. Osteoblastic lineage markers were studied using immunocytochemistry and RT‐PCR. Staminality markers were also analysed using flow cytometry, magnetic cell separation technology, immunocytochemistry and RT‐PCR. Starting from p2, MSK and control cells formed nodules with a behaviour similar to that of calcifying pericytes; however, Ca2PO4 was only found in the MSK cultures. The MSK cells had morphologies and immunophenotypes resembling those of pericytes or stromal stem cells and were positive for vimentin, ZO1, αSMA and CD146. In addition, the MSK cells expressed osteocalcin and osteonectin, indicating an osteoblast‐like phenotype. In contrast to the control cells, GDNF was down‐regulated in the MSK cells. Stable GDNF knockdown was established in the HK2 cell line and was found to promote Ca2PO4 deposition when the cells were incubated with calcifying medium by regulating the osteonectin/osteopontin ratio in favour of osteonectin. Our data indicate that the human papilla may be a perivascular niche in which pericyte/stromal‐like cells can undergo osteogenic differentiation under particular conditions and suggest that GDNF down‐regulation may have influenced the observed phenomenon.

Novel mutations of the CLCN5 gene including a complex allele and A 5′ UTR mutation in Dent disease 1

To the Editor : Dent disease (DD) 1 (OMIM 300009) is an X-linked disorder of renal tubular function, characterized by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis and progressive renal failure, and associated with mutations in the ClC-5 Cl−/H+ endosomal exchanger all located in the coding region of the CLCN5 gene (1, 2). We report here the identification of 15 novel mutations including a complex allele and a nucleotide substitution in the 5′UTR region of the CLCN5 gene. Thirty patients with clinical suspicion of DD were screened by single-strand conformation polymorphism (SSCP) analysis and direct sequencing for the presence of mutations in the coding sequence and the exon-intron boundaries as well as in the 5′untranslated exons of the CLCN5 gene. All the patients were of Italian origin, except 1 from the United Kingdom. Sixteen of the 30 patients presented CLCN5 mutations of which 15 were novel and 1 recurrent (S244L). The phenotypic characteristics of patients with and without CLCN5 mutations are reported in Table 1. The table shows that LMWP, hypercalciuria and nephrocalcinosis, represent the triad of manifestations most relevant for the diagnosis of DD 1. Table 2 reports the 15 novel CLCN5 mutations identified in DD patients. Each of the missense and deletion–insertion mutations predicts a structurally significant alteration to the ClC-5 Cl−/H+ exchanger, and is thus likely to be of importance in the aetiology of the disease. The three frameshift mutations, leading to premature termination codons (PTC), can induce either a truncated ClC-5 product or mRNA degradation through nonsense-mediated decay which are likely to result in a loss of antiporter function and chloride conductance. The two in-frame codon deletion and the insertion are thought to alter the ClC-5 α-helices H and I, which are two of the four major helices involved in the formation of dimer interface (3). All the missense mutations, except one located in the C-terminal domain, are at or near the ClC-5 dimer interface. Moreover they involved amino acids conserved among different species or present in all known ClCs and were predicted with a high confidence to affect protein function by both the web program SIFT (4) and PolyPhen (5). For four out of six missense mutations (W58L, G512D, V519D, P621L) and for T277_L278 ins S mutation it was possible to establish that they segregated with DD trait in family members. Three intronic mutations affecting the canonical consensus splicing donor site of intron 4, 8 and 11 were detected in three patients. In two of them, the presence of an aberrantly spliced ClC-5 mRNA in leukocytes, leading to a truncated or absent protein, confirmed the functional significance of the mutations (Fig. 1). The presence of an aberrantly spliced mRNA due to the IVS4 +4 A>G could not be proved by transcription polymerase chain reaction (RT-PCR) analysis because the patient’s RNA was not available. The Automated Splice-Site Analysis program (ASSA; (6)), however, predicts that this variant would lower the strength of the donor splice-site leading probably to exon 4 skipping. We identified a complex allele (S386F and S388fsX434) in one patient from North Italy, inherited from his mother and grandmother. Since both the web program SIFT and PolyPhen predicted that the missense mutation S386F did not affect protein function, and the search for potential exonic splicing enhancer (ESE) motifs using ESE finder (7), RESCUE-ESE (8) and ASSA did not evidence any modification, we wondered if the mutation was indeed a polymorphism. The substitution was not found in our series of 69 patients affected by DD and in 311 X chromosomes from umbilical cord DNA samples thus