Mutational signatures in GATA3 transcription factor and its DNA binding domain that stimulate breast cancer and HDR syndrome.

Transcription factors (TFs) play important roles in many biochemical processes. Many human genetic disorders have been associated with mutations in the genes encoding these transcription factors, and so those mutations became targets for medications and drug design. In parallel, since many transcription factors act either as tumor suppressors or oncogenes, their mutations are mostly associated with cancer. In this perspective, we studied the GATA3 transcription factor when bound to DNA in a crystal structure and assessed the effect of different mutations encountered in patients with different diseases and phenotypes. We generated all missense mutants of GATA3 protein and DNA within the adjacent and the opposite GATA3:DNA complex models. We mutated every amino acid and studied the new binding of the complex after each mutation. Similarly, we did for every DNA base. We applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations. After analyzing our data, we identified amino acids and DNA bases keys for binding. Furthermore, we validated those findings against experimental genetic data. Our results are the first to propose in silico modeling for GATA:DNA bound complexes that could be used to score effects of missense mutations in other classes of transcription factors involved in common and genetic diseases.

The structural and functional workings of KEOPS.

KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) is a five-subunit protein complex that is highly conserved in eukaryotes and archaea and is essential for the fitness of cells and for animal development. In humans, mutations in KEOPS genes underlie Galloway-Mowat syndrome, which manifests in severe microcephaly and renal dysfunction that lead to childhood death. The Kae1 subunit of KEOPS catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine (t6A), while the auxiliary subunits Cgi121, the kinase/ATPase Bud32, Pcc1 and Gon7 play a supporting role. Kae1 orthologs are also present in bacteria and mitochondria but function in distinct complexes with proteins that are not related in structure or function to the auxiliary subunits of KEOPS. Over the past 15 years since its discovery, extensive study in the KEOPS field has provided many answers towards understanding the roles that KEOPS plays in cells and in human disease and how KEOPS carries out these functions. In this review, we provide an overview into recent advances in the study of KEOPS and illuminate exciting future directions.

First patient with ILNEB syndrome due to pathogenic variants in ITGA3 surviving to adulthood.

Interstitial Lung disease, Nephrotic syndrome and Epidermolysis Bullosa, also referred to as ILNEB syndrome is an extremely rare autosomal recessive condition, caused by pathogenic variants in ITGA3. 11 patients have previously been diagnosed with ILNEB syndrome of whom 7 died in infancy or early childhood. We report the only patient with ILNEB syndrome who survived past adolescence, partly due to a double lung transplant. Additionally, our patient showed oral, nasal and gynecological symptoms not previously reported in patients with ILNEB syndrome.

Cholemic Nephrosis: An Autopsy Study of a Forgotten Entity.

OBJECTIVE: The aim of the study is to do a clinicopathologic study of post mortem kidney biopsies with significant deposition of bilirubin pigment within tubular epithelial cells and in the lumen of distal tubules as a bile cast. MATERIAL AND METHOD: All post mortem specimens with acute tubular necrosis, with the presence of bile casts in tubules or bile pigment deposition in the tubular epithelium during the period 2015-2018 were examined for gross and histopathology along with biochemical parameters and viral markers. RESULTS: Bile casts with sloughed renal tubular epithelial cells and occasional macrophages were present in the distal convoluted tubule in 78.6% of biopsies (11/14). The plugging of distal convoluted tubule with casts was similar to that seen in myeloma and myoglobin cast nephropathies. Bilirubin pigment deposition was present in 35.7% (5/14) of cases. The frequency of bile casts in each biopsy was variable and it did not have any association with serum bilirubin levels or etiology of liver dysfunction. A striking difference from earlier studies is the high number of toxin-induced liver damage including six cases of paraquat and 2 cases of yellow phosphorus poisoning. CONCLUSION: This study proves importance of the bile cast nephropathy as a reason for kidney injury, especially with varied hepatotoxic etiologies, especially paraquat and yellow phosphorus.

Genetic ablation of SLK exacerbates glomerular injury in adriamycin nephrosis in mice.

Ste20-like kinase SLK is critical for embryonic development and may play an important role in wound healing, muscle homeostasis, cell migration, and tumor growth. Mice with podocyte-specific deletion of SLK show albuminuria and damage to podocytes as they age. The present study addressed the role of SLK in glomerular injury. We induced adriamycin nephrosis in 3- to 4-mo-old control and podocyte SLK knockout (KO) mice. Compared with control, SLK deletion exacerbated albuminuria and loss of podocytes, synaptopodin, and podocalyxin. Glomeruli of adriamycin-treated SLK KO mice showed diffuse increases in the matrix and sclerosis as well as collapse of the actin cytoskeleton. SLK can phosphorylate ezrin. The complex of phospho-ezrin, Na+/H+ exchanger regulatory factor 2, and podocalyxin in the apical domain of the podocyte is a key determinant of normal podocyte architecture. Deletion of SLK reduced glomerular ezrin and ezrin phosphorylation in adriamycin nephrosis. Also, deletion of SLK reduced the colocalization of ezrin and podocalyxin in the glomerulus. Cultured glomerular epithelial cells with KO of SLK showed reduced ezrin phosphorylation and podocalyxin expression as well as reduced F-actin. Thus, SLK deletion leads to podocyte injury as mice age and exacerbates injury in adriamycin nephrosis. The mechanism may at least in part involve ezrin phosphorylation as well as disruption of the cytoskeleton and podocyte apical membrane structure.

Novel heterozygous GATA3 and SLC34A3 variants in a 6-year-old boy with Barakat syndrome and hypercalciuria.

BACKGROUND: Barakat syndrome is an autosomal dominant disorder characterized by the triad of hypoparathyroidism, sensorineural deafness, and renal anomalies and is caused by mutations in GATA3 gene. SLC34A3 is the cause gene of hypophosphatemic rickets with hypercalciuria, and heterozygous carriers may have milder clinical symptoms. The aim of this study was to identify the underlying genetic cause of a patient who initially presented with renal failure, hypercalciuria, kidney stone, and bilateral sensorineural deafness. METHODS: A 6-year-old boy with complex clinical presentations was investigated. Comprehensive medical evaluations were performed including auditory function tests, endocrine function tests, metabolic studies, and imaging examinations. Molecular diagnoses were analyzed by trio whole-exome sequencing. RESULTS: One novel de novo deleterious variant (c. 324del) of the GATA3 gene was identified in the patient. The patient can be diagnosed with Barakat syndrome. In addition, one novel variant (c. 589A>G) of the SLC34A3 gene was detected, which was inherited from the father. This heterozygous variant can explain the hypercalciuria and kidney stone that occurred in both the patient and his father. CONCLUSION: This study provides a special case which is phenotype-driven dual diagnoses, and the two novel variants can parsimoniously explain the complex clinical presentations of this patient.

Genetic Ablation of Calcium-independent Phospholipase A2γ Exacerbates Glomerular Injury in Adriamycin Nephrosis in Mice.

Genetic ablation of calcium-independent phospholipase A2γ (iPLA2γ) in mice results in marked damage of mitochondria and enhanced autophagy in glomerular visceral epithelial cells (GECs) or podocytes. The present study addresses the role of iPLA2γ in glomerular injury. In adriamycin nephrosis, deletion of iPLA2γ exacerbated albuminuria and reduced podocyte number. Glomerular LC3-II increased and p62 decreased in adriamycin-treated iPLA2γ knockout (KO) mice, compared with treated control, in keeping with increased autophagy in KO. iPLA2γ KO GECs in culture also demonstrated increased autophagy, compared with control GECs. iPLA2γ KO GECs showed a reduced oxygen consumption rate and increased phosphorylation of AMP kinase (pAMPK), consistent with mitochondrial dysfunction. Adriamycin further stimulated pAMPK and autophagy. After co-transfection of GECs with mito-YFP (to label mitochondria) and RFP-LC3 (to label autophagosomes), or RFP-LAMP1 (to label lysosomes), there was greater colocalization of mito-YFP with RFP-LC3-II and with RFP-LAMP1 in iPLA2γ KO GECs, compared with WT, indicating enhanced mitophagy in KO. Adriamycin increased mitophagy in WT cells. Thus, iPLA2γ has a cytoprotective function in the normal glomerulus and in glomerulopathy, as deletion of iPLA2γ leads to mitochondrial damage and impaired energy homeostasis, as well as autophagy and mitophagy.

Estrogen-related receptor-α mediates puromycin aminonucleoside-induced mesangial cell apoptosis and inflammatory injury.

Glomerular diseases are the leading cause of chronic kidney disease, and mesangial cells (MCs) have been demonstrated to be involved in the pathogenesis. Puromycin aminonucleoside (PAN) is a nephrotoxic drug that induces glomerular injury with elusive mechanisms. The present study was undertaken to investigate the role of PAN in MC apoptosis, as well as the underlying mechanism. Here we found that PAN induced MC apoptosis accompanied by declined cell viability and enhanced inflammatory response. The apoptosis was further evidenced by increments of apoptosis regulator BAX (BAX) and caspase-3 expression. In line with the apoptotic response in MCs following PAN treatment, we also found a remarkable induction of estrogen-related receptor-α (ERRα), an orphan nuclear receptor, at both mRNA and protein levels. Interestingly, ERRα silencing by an siRNA approach resulted in an attenuation of the apoptosis and inflammatory response caused by PAN. More importantly, overexpression of ERRα in MCs significantly triggered MC apoptosis in line with increased BAX and caspase-3 expression. In PAN-treated MCs, ERRα overexpression further aggravated PAN-induced apoptosis. In agreement with the in vitro study, we also observed increased ERRα expression in line with enhanced apoptotic response in renal cortex from PAN-treated rats. These data suggest a detrimental effect of ERRα on PAN-induced MC apoptosis and inflammatory response, which could help us to better understand the pathogenic mechanism of MC injury in PAN nephropathy.

WDR73-related galloway mowat syndrome with collapsing glomerulopathy.

Galloway-Mowat syndrome (GAMOS [MIM 251300]) is a rare autosomal recessive disorder that manifests as a combination of nephrotic syndrome, brain abnormalities and developmental delay. It is a clinically and genetically heterogeneous disease. The WDR73 variations are associated with GAMOS1. Here we report two consanguineous families affected by GAMOS1. In the first family, three sisters are affected and in the second family, only one index case is identified. They all show a nephrotic syndrome, a neurological involvement and a collapsing glomerulopathy. The analysis of mutations of WDR73 revealed a new homozygous missense mutation NM_032856.3 c.293T > C; p.(Leu98Pro) in two patients from the first family, and a new homozygous missense mutation NM_032856.3: c.767G > A; p.(Arg256Gln) in the second one. This study extended the clinical and molecular spectrum of GAMOS1 with other cases associated with collapsing glomerulopathy and two novel WDR73 variations that are most likely pathogenic.

NIX-mediated mitophagy protects against proteinuria-induced tubular cell apoptosis and renal injury.

Proteinuria, the most common symptom of renal injury, is an independent factor for renal tubular injury. However, the underlying mechanism remains to be fully elucidated. Mitochondrion is an important target for proteinuria-induced renal tubular cell injury. Insufficient mitophagy exacerbates cell injury by initiating mitochondrial dysfunction-related cell apoptosis. In the experiment, the role of NIP3-like protein X (NIX)-mediated mitophagy was investigated in proteinuria-induced renal injury. In this study, we demonstrated that NIX expression was reduced in renal tubules and correlated with the decline of estimated glomerular filtration rate and increase of the proteinuria in patients. In proteinuric mice, NIX-mediated mitophagy was significantly suppressed. Meanwhile, the proteinuric mice exhibited renal dysfunction, increased mitochondrial fragmentation, and tubular cell apoptosis. Overexpression of NIX attenuated those disruptions in proteinuric mice. In cultured renal tubular epithelial cells, albumin induced a decrease in NIX-mediated mitophagy and an increase in cell apoptosis. Overexpression of NIX attenuated albumin-induced cell apoptosis, whereas NIX siRNA aggravated these perturbations. These results indicate that proteinuria suppresses NIX-mediated mitophagy in the renal tubular epithelial cell, which triggers the cell undergoing mitochondria-dependent cell apoptosis. Collectively, our finding suggests that restoration of NIX-mediated mitophagy might be a novel therapeutic target for alleviating proteinuria-induced kidney injury.

Morphological Processes of Foot Process Effacement in Puromycin Aminonucleoside Nephrosis Revealed by FIB/SEM Tomography.

BACKGROUND: Foot process effacement is one of the pathologic indicators of podocyte injury. However, the morphologic changes associated with it remain unclear. METHODS: To clarify the developmental process, we analyzed puromycin nephrotic podocytes reconstructed from serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. RESULTS: Intact podocytes consisted of four subcellular compartments: cell body, primary process, ridge-like prominence (RLP), and foot process. The RLP, a longitudinal protrusion from the basal surface of the cell body and primary process, served as an adhesive apparatus for the cell body and primary process to attach to the glomerular basement membrane. Foot processes protruded from both sides of the RLP. In puromycin nephrotic podocytes, foot process effacement occurred in two ways: by type-1 retraction, where the foot processes retracted while maintaining their rounded tips; or type-2 retraction, where they narrowed across their entire lengths, tapering toward the tips. Puromycin nephrotic podocytes also exhibited several alterations associated with foot process effacement, such as deformation of the cell body, retraction of RLPs, and cytoplasmic fragmentation. Finally, podocytes were reorganized into a broad, flattened shape. CONCLUSIONS: The three-dimensional reconstruction of podocytes by serial FIB/SEM images revealed the morphologic changes involved in foot process effacement in greater detail than previously described.

Homozygous splicing mutation in NUP133 causes Galloway-Mowat syndrome.

OBJECTIVE: Galloway-Mowat syndrome (GAMOS) is a neural and renal disorder, characterized by microcephaly, brain anomalies, and early onset nephrotic syndrome. Biallelic mutations in WDR73 and the 4 subunit genes of the KEOPS complex are reported to cause GAMOS. Furthermore, an identical homozygous NUP107 (nucleoporin 107kDa) mutation was identified in 4 GAMOS-like families, although biallelic NUP107 mutations were originally identified in steroid-resistant nephrotic syndrome. NUP107 and NUP133 (nucleoporin 133kDa) are interacting subunits of the nuclear pore complex in the nuclear envelope during interphase, and these proteins are also involved in centrosome positioning and spindle assembly during mitosis. METHODS: Linkage analysis and whole exome sequencing were performed in a previously reported GAMOS family with brain atrophy and steroid-resistant nephrotic syndrome. RESULTS: We identified a homozygous NUP133 mutation, c.3335-11T>A, which results in the insertion of 9bp of intronic sequence between exons 25 and 26 in the mutant transcript. NUP133 and NUP107 interaction was impaired by the NUP133 mutation based on an immunoprecipitation assay. Importantly, focal cortical dysplasia type IIa was recognized in the brain of an autopsied patient and focal segmental glomerulosclerosis was confirmed in the kidneys of the 3 examined patients. A nup133-knockdown zebrafish model exhibited microcephaly, fewer neuronal cells, underdeveloped glomeruli, and fusion of the foot processes of the podocytes, which mimicked human GAMOS features. nup133 morphants could be rescued by human wild-type NUP133 mRNA but not by mutant mRNA. INTERPRETATION: These data indicate that the biallelic NUP133 loss-of-function mutation causes GAMOS. Ann Neurol 2018;84:814-828.

In glomerular cells of puromycin aminonucleoside nephrosis rats both phosphorylated and total STAT3 levels increased during proteinuria.

Recent studies showed that JAK/STAT pathway plays role in glomerular damages. The fact that STAT3 could be activated also by oxidative stress make Puromycin Aminonucleoside (PAN) Nephrosis model very appropriate for examination of STAT3 expression changes in glomerular pathology. Along with a control group, three PAN groups sacrificed on different days were formed by the i.p. injection of PAN for 5 consecutive days. Throughout the experiment, 24-hour-urines were collected on specific days and proteinuria levels were monitored. At the end of the experiments, tissue specimens were stained immunohistochemically for both total and phosphorylated STAT3 and evaluated subjectively. They were also examined ultrastructurally in transmission electron microscope. The proteinuria levels did not increase significantly on 5th day but showed a dramatic increase on 10th and 15th days. On 20th and 25th days, urinary protein levels gradually decreased. Ultrastructural examinations showed glomerular damages such as significant decrease in slit pore number, a significant gradual increase in glomerular basement membrane thickness and podocyte hypertrophy on 5th and 15th days; besides significant increase in mesangial matrix. The first significant increases in phosphorylated and total STAT3 levels occurred in 5th day and 15th day groups respectively. These increases diminished in 25th day group. Regarding all the findings, it was deduced that STAT3 is one of the active factors in glomerular pathologies.

Mechanisms Underlying Exacerbation of Osmotic Nephrosis Caused by Pre-existing Kidney Injury.

Osmotic nephrosis, a disease caused by intravenous infusion of various fluids such as hypertonic sucrose and isotonic polysaccharide-based plasma volume expanders, exhibits specific histopathological features, including vacuolated and swollen proximal tubules, ie, "clear tubules". Pre-existing kidney injury exacerbates this condition, resulting in major clinical problems. However, the underlying mechanisms are unclear. Animal models often yield results that are directly translatable to humans. Therefore, in this study, we performed detailed histopathological analyses of the formation of clear tubules in rats treated with gentamicin or ischemia/reperfusion (IR) operation followed by dextran administration. The results showed that clear tubules may originate from regenerative tubules. Additionally, we classified regenerative tubules into 3 categories based on their development, with a particular focus on the middle and late stages. Comprehensive microarray and real-time polymerase chain reaction analyses of mRNA extracted from regenerative tubules at each stage using laser microdissection revealed that regenerative tubules in the middle stage showed an imbalance between dextran absorption and metabolism, resulting in accumulation of dextran, particularly in the cytoplasm of the tubules. Overall, our findings demonstrated that clear tubules originated from regenerated tubules and that tubules at the middle stage became clear tubules because of an imbalance during their development. This could explain why osmotic nephrosis is exacerbated in the presence of kidney lesions.

Trpc6 inactivation confers protection in a model of severe nephrosis in rats.

Mutations in canonical transient receptor potential-6 (TRPC6) channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined a possible role for TRPC6 in the progression of chronic puromycin aminonucleoside (PAN) nephrosis in Sprague-Dawley rats, a classic model of acquired nephrotic syndromes. We used CRISPR/Cas9 technology to delete a 239-bp region within exon 2 of the Trpc6 gene (Trpc6del allele). Trpc6del/del rats expressed detectable Trpc6 transcripts missing exon 2, and TRPC6 proteins could be detected by immunoblot of renal cortex. However, the abundance of Trpc6 transcripts and TRPC6 protein in renal cortex was much lower than in Trpc6wt/wt littermates, and functional TRPC6 channels could not be detected in whole-cell recordings from glomerular cells cultured from Trpc6del/del animals, possibly because of disruption of ankyrin repeats 1 and 2. During the chronic phase of PAN nephrosis, Trpc6del/del rats had reduced urine albumin excretion, reduced serum cholesterol and triglycerides, and improved azotemia compared to wild-type Trpc6wt/wt littermates. Glomerulosclerosis was severe during chronic PAN nephrosis in Trpc6wt/wt rats but was markedly reduced in Trpc6del/del littermates. Trpc6del/del animals also had less severe tubulointerstitial fibrosis as assessed by several biochemical and histological analyses, as well as reduced foot process effacement and glomerular basement thickening compared to Trpc6wtt/wt controls. None of the manipulations in this study affected the abundance of TRPC5 channels in renal cortex. TRPC3 was increased in PAN nephrosis and in Trpc6del/del rats. These data support a role for TRPC6 channels in driving an acquired form of secondary FSGS. KEY MESSAGES: We examined aminonucleoside nephrosis in rats with wild type and inactivated TRPC6. TRPC6 channels were inactivated by CRISPR/Cas9 editing of the Trpc6 gene. TRPC6 inactivation reduced albuminuria in the chronic but not the acute phase. TRPC6 inactivation reduced glomerulosclerosis and ultrastructural changes. TRPC6 inactivation also reduced interstitial changes and renal fibrosis.

Hemicentin 1 influences podocyte dynamic changes in glomerular diseases.

Different complex mechanisms control the morphology of podocyte foot processes and their interactions with the underlying basement membrane. Injuries to this system often cause glomerular dysfunction and albuminuria. The present study aimed at identifying early markers of glomerular damage in diabetic nephropathy. For this purpose, we performed a microarray analysis on kidneys of 3-wk-old peroxisome proliferator-activated receptor-γ (PPARγ)-null and AZIP/F1 mice, which are two models of diabetic nephropathy due to lipodystrophy. This was followed by functional annotation of the enriched clusters of genes. One of the significant changes in the early stages of glomerular damage was the increase of hemicentin 1 (HMCN1). Its expression and distribution were then studied by real-time PCR and immunofluorescence in various models of glomerular damage and on podocyte cell cultures. HMCN1 progressively increased in the glomeruli of diabetic mice, according to disease severity, as well as in puromycin aminonucleoside (PA)-treated rats. Studies on murine and human podocytes showed an increased HMCN1 deposition upon different pathological stimuli, such as hyperglycemia, transforming growth factor-ß (TGF-ß), and PA. In vitro silencing studies showed that HMCN1 mediated the rearrangements of podocyte cytoskeleton induced by TGF-ß. Finally, we demonstrated an increased expression of HMCN1 in the kidneys of patients with proteinuric nephropathies. In summary, our studies identified HMCN1 as a new molecule involved in the dynamic changes of podocyte foot processes. Its increased expression associated with podocyte dysfunction points to HMCN1 as a possible marker for the early glomerular damage occurring in different proteinuric nephropathies.

Rac1 in podocytes promotes glomerular repair and limits the formation of sclerosis.

Rac1, a Rho family member, is ubiquitously expressed and participates in various biological processes. Rac1 expression is induced early in podocyte injury, but its role in repair is unclear. To investigate the role of Rac1 expression in podocytes under pathological conditions, we used podocyte-specific Rac1 conditional knock-out (cKO) mice administered adriamycin (ADR), which causes nephrosis and glomerulosclerosis. Larger areas of detached podocytes, more adhesion of the GBM to Bowman's capsule, and a higher ratio of sclerotic glomeruli were observed in Rac1 cKO mice than in control mice, whereas no differences were observed in glomerular podocyte numbers in both groups after ADR treatment. The mammalian target of rapamycin (mTOR) pathway, which regulates the cell size, was more strongly suppressed in the podocytes of Rac1 cKO mice than in those of control mice under pathological conditions. In accordance with this result, the volumes of podocytes in Rac1 cKO mice were significantly reduced compared with those of control mice. Experiments using in vitro ADR-administered Rac1 knockdown podocytes also supported that a reduction in Rac1 suppressed mTOR activity in injured podocytes. Taken together, these data indicate that Rac1-associated mTOR activation in podocytes plays an important role in preventing the kidneys from developing glomerulosclerosis.

Pathological and molecular studies of the renal trematode Paratanaisia bragai in Indian peafowls (Pavo cristatus).

Endoparasitic diseases are commonly encountered in free-ranging birds. Although not all endoparasites cause disease, persistent infection with large numbers of parasites almost always affects normal physiological functions, leading to deleterious effects on the host. This paper describes the anatomopathological alterations caused by the renal trematode Paratanaisia bragai in Indian peafowl (n = 3) and examines the phylogeny of these and related parasites. Peafowl from forests in and around the Bareilly region, Uttar Pradesh, India, were necropsied, and microscopic and molecular investigations were performed. The peafowl were confirmed to be infected with P. bragai. Significant gross pathological lesions suggested nephrosis, and microscopic findings indicated a mild-to-moderate degree of nephrosis caused by the parasites in the tissue. The parasites were identified as P. bragai by histomorphological analysis of adult and eggs in the ureters, and the identification was confirmed by PCR and phylogenetic analysis. Nucleotide sequencing of the PCR products from the renal trematodes recovered from Indian peafowl revealed a close association with P. bragai from Columbiformes in the United Kingdom and Spain. The pathology and molecular epidemiology of parasitic diseases affecting peafowl is not well understood in India. This is the first report from India and the second report worldwide to document P. bragai infection in peafowl.

Pathology and Epidemiology of Oxalate Nephrosis in Cheetahs.

To investigate cases of acute oxalate nephrosis without evidence of ethylene glycol exposure, archived data and tissues from cheetahs ( Acinonyx jubatus) from North America ( n = 297), southern Africa ( n = 257), and France ( n = 40) were evaluated. Renal and gastrointestinal tract lesions were characterized in a subset of animals with ( n = 100) and without ( n = 165) oxalate crystals at death. Crystals were confirmed as calcium oxalate by Raman spectroscopy in 45 of 47 cheetahs tested. Crystals were present in cheetahs from 3.7 months to 15.9 years old. Cheetahs younger than 1.5 years were less likely to have oxalates than older cheetahs ( P = .034), but young cheetahs with oxalates had more oxalate crystals than older cheetahs ( P < .001). Cheetahs with oxalate crystals were more likely to have renal amyloidosis, interstitial nephritis, or colitis and less likely to have glomerular loop thickening or gastritis than those without oxalates. Crystal number was positively associated with renal tubular necrosis ( P ≤ .001), regeneration ( P = .015), and casts ( P ≤ .001) but inversely associated with glomerulosclerosis, renal amyloidosis, and interstitial nephritis. Crystal number was unrelated to the presence or absence of colitis and was lower in southern African than American and European animals ( P = .01). This study found no evidence that coexisting chronic renal disease (amyloidosis, interstitial nephritis, or glomerulosclerosis), veno-occlusive disease, gastritis, or enterocolitis contributed significantly to oxalate nephrosis. Oxalate-related renal disease should be considered as a potential cause of acute renal failure, especially in young captive cheetahs. The role of location, diet, stress, and genetic predisposition in the pathogenesis of oxalate nephrosis in cheetahs warrants further study.

Re-expression of Sall1 in podocytes protects against adriamycin-induced nephrosis.

The highly conserved spalt (sal) gene family members encode proteins characterized by multiple double zinc finger motifs of the C2H2 type. Humans and mice each have four known Sal-like genes (SALL1-4 in humans and Sall1-4 in mice). Sall1 is known to have a crucial role in kidney development. To explore the significance of Sall1 in differentiated podocytes, we investigated podocyte-specific Sall1-deficient mice (Sall1 KOp°d°/p°d°) using a podocin-Cre/loxP system and siRNA Sall1 knockdown (Sall1 KD) podocytes. Under physiological conditions, Sall1 KOp°d°/p°d° mice exhibited no proteinuria during their lifetime, but foot-process effacement was detected in some of the podocytes. To elucidate the role of Sall1 in injured podocytes, we used an adriamycin (ADR)-induced model of nephrosis and glomerulosclerosis. Surprisingly, the expression of Sall1 was elevated in control mice on day 14 after ADR injection. On day 28 after ADR injection, Sall1 KOp°d°/p°d° mice exhibited significantly higher levels of proteinuria and higher numbers of sclerotic glomeruli. Differentiated Sall1 KD podocytes showed a loss of synaptopodin, suppressed stress fiber formation, and, ultimately, impaired directed cell migration. In addition, the loss of Sall1 increased the number of apoptotic podocytes following ADR treatment. These results indicated that Sall1 has a protective role in podocytes; thus, we investigated the endoplasmic reticulum stress marker GRP78. GRP78 expression was higher in ADR-treated Sall1 KOp°d°/p°d° mice than in control mice. Sall1 appeared to influence the expression of GRP78 in injured podocytes. These results suggest that Sall1 is associated with actin reorganization, endoplasmic reticulum stress, and apoptosis in injured podocytes. These protective aspects of Sall1 re-expression in injured podocytes may have the potential to reduce apoptosis and possibly glomerulosclerosis.