Characterization of temporospatial distribution of renal tubular casts by nephron tracking after ischemia-reperfusion injury.

Renal tubular casts originating from detached epithelial cells after ischemia-reperfusion injury (IRI) can obstruct tubules and negatively impact glomerular filtration rate. Using multiphoton imaging of 400-µm-thick kidney sections, the distribution of casts and morphometric measurement of tubules was performed along the entire nephron for the first time. Tubular nuclei are shed before cell detachment, and visually occlusive casts (grade 3) appeared at 12 h after IRI at the S3/thin descending limb (tDL) junction. Grade 3 casts peaked at 24 h after injury [present in 99% of S3, 78% of tDL, 76% of thin ascending limb (tAL), 60% of medullary thick ascending limb (mTAL), and 10% of connecting tubule segments]. Cast formation in the S3 correlated with selective loss of cell numbers from this tubule segment. By day 3, most mTALs and connecting tubules were cast free, whereas 72% of S3 tubules and 58% of tDLs still contained grade 3 casts. Although bulk phagocytosis of cast material by surviving tubular cells was not observed, mass spectrometry identified large numbers of tryptic peptides in the outer medulla, and trypsin levels were significantly increased in the kidney and urine 24 h after IRI. Administration of either antipain or camostat to inhibit trypsin extended cast burden to the S2, led to sustained accumulation of S3 casts after IRI, but did not affect cast burden in the mTAL or renal function. Our data provide detailed and dynamic mapping of tubular cast formation and resolution after IRI that can inform future interventions to accelerate cast clearance and renal recovery.NEW & NOTEWORTHY This detailed characterization of the dynamic distribution of dead cell debris in ischemically injured kidney tubules reveals which cells in the kidney are most severely injured, when and where tubular casts form, and when (and to a lesser extent, how) they are cleared.

The Terminator mouse is a diphtheria toxin-receptor knock-in mouse strain for rapid and efficient enrichment of desired cell lineages.

Biomedical research often requires primary cultures of specific cell types, which are challenging to obtain at high purity in a reproducible manner. Here we engineered the murine Rosa26 locus by introducing the diphtheria toxin receptor flanked by loxP sites. The resultant strain was nicknamed the Terminator mouse. This approach results in diphtheria toxin-receptor expression in all non-Cre expressing cell types, making these cells susceptible to diphtheria toxin exposure. In primary cultures of kidney cells derived from the Terminator mouse, over 99.99% of cells were dead within 72 h of diphtheria toxin treatment. After crossing the Terminator with the podocin-Cre (podocyte specific) mouse or the Ggt-Cre (proximal tubule specific) mouse, diphtheria toxin treatment killed non-Cre expressing cells but spared podocytes and proximal tubule cells, respectively, enriching the primary cultures to over 99% purity, based on both western blotting and immunostaining of marker proteins. Thus, the Terminator mouse can be a useful tool to selectively and reproducibly obtain even low-abundant cell types at high quantity and purity.

Brg1 determines urothelial cell fate during ureter development.

Developing and adult ureters express the epigenetic regulator Brg1, but the role of Brg1 in ureter development is not well understood. We conditionally ablated Brg1 in the developing ureter using Hoxb7-Cre and found that Brg1 expression is upstream of p63, Pparγ, and sonic hedgehog (Shh) expression in the ureteral epithelium. In addition, epithelial stratification in the basal cells required Brg1-dependent p63 expression, whereas terminal differentiation of the umbrella cells required Brg1-dependent Pparγ expression. Furthermore, the loss of ureteric Brg1 resulted in failure of Shh expression, which correlated with reduced smooth muscle cell development and hydroureter. Taken together, we conclude that Brg1 expression unifies three aspects of ureter development: maintenance of the basal cell population, guidance for terminal differentiation of urothelial cells, and proper investment of ureteral smooth muscle cells.

Genetic or pharmacologic blockade of EGFR inhibits renal fibrosis.

Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of α-smooth muscle actin (α-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-ß1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin-2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-ß1- or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of α-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.

Increased tubular proliferation as an adaptive response to glomerular albuminuria.

Renal tubular atrophy accompanies many proteinuric renal diseases, suggesting that glomerular proteinuria injures the tubules. However, local or systemic inflammation and filtration of abnormal proteins known to directly injure tubules are also present in many of these diseases and animal models; therefore, whether glomerular proteinuria directly causes tubular injury is unknown. Here, we examined the renal response to proteinuria induced by selective podocyte loss. We generated mice that express the diphtheria toxin receptor exclusively in podocytes, allowing reproducible dose-dependent, specific ablation of podocytes by administering diphtheria toxin. Ablation of <20% of podocytes resulted in profound albuminuria that resolved over 1-2 weeks after the re-establishment of normal podocyte morphology. Immediately after the onset of albuminuria, proximal tubule cells underwent a transient burst of proliferation without evidence of tubular damage or increased apoptosis, resulting in an increase in total tubular cell numbers. The proliferative response coincided with detection of the growth factor Gas6 in the urine and phosphorylation of the Gas6 receptor Axl in the apical membrane of renal tubular cells. In contrast, ablation of >40% of podocytes led to progressive glomerulosclerosis, profound tubular injury, and renal failure. These data suggest that glomerular proteinuria in the absence of severe structural glomerular injury activates tubular proliferation, potentially as an adaptive response to minimize the loss of filtered proteins.

Cellular maintenance and repair of the kidney.

The mammalian kidney is a highly complex organ that requires the precise structural arrangement of multiple cell types for effective function. The need to filter large volumes of plasma at the glomerulus followed by active reabsorption of nearly 99% of that filtrate by the tubules creates vulnerability in both compartments for cell injury. Thus maintenance of cell viability and replacement of those cells that are lost are essential for functional stability of the kidney. This review addresses our current understanding of how cells from the glomerular, tubular, and interstitial compartments arise during development and the manner in which they may be regenerated in the adult organ. In addition, we discuss the data regarding the role of organ-specific and bone marrow-derived stem and progenitor cells in the replacement/repair process, as well as the potential for ex vivo programming of stem cells toward a renal lineage.

The commonly used beta-actin-GFP transgenic mouse strain develops a distinct type of glomerulosclerosis.

Green fluorescent protein (GFP) transgenic animals are widely used in biomedical research. We observed that the commonly used beta-actin-GFP transgenic mouse has renal defects with proteinuria starting as early as 5 weeks of age. Histological analysis reveals a widespread increase in glomerular extracellular matrix, occasional mesangiolysis, and secondary tubulointerstitial injury. Electron microscopic (EM) analysis reveals dramatic thickening of the glomerular basement membrane (GBM). Several other transgenic strains with GFP on ubiquitous promoters including beta-actin (with insertion in a different location) and ubiquitin C show no renal abnormalities. Western blot analysis on crude glomerular preparations from several GFP transgenic strains revealed that higher levels of GFP expression might be responsible for the observed pathogenesis. Mapping of the transgene insertion site by inverse PCR indicates that the beta-actin GFP transgene does not cause insertional mutagenesis nor does it modify the transcription level of adjacent genes. Taken together, this strain of beta-actin-GFP transgenic mouse may be used to study the mechanism of GBM expansion. Moreover, experiments using this strain of GFP mouse should be hereafter carefully planned because its renal pathology may interfere with data interpretation.

An inducible mouse model for PAX2-dependent glomerular disease: insights into a complex pathogenesis.

Pax2 is a transcription factor with important functions during kidney development . Ectopic expression of Pax2 in podocytes has been reported in various glomerular diseases , but the functional relevance remains unknown. We developed an inducible mouse model that allows activation of Pax2 specifically in podocytes. Persistent expression of Pax2 did not interfere with the initial differentiation of podocytes, but mice ectopically expressing PAX2 developed end-stage renal failure soon after birth. Similarly, activation of PAX2 in healthy adult animals resulted in renal disease within 3 weeks after podocyte-specific induction of a deleter Cre. PAX2 activation caused repression of the podocyte key regulator molecule Wt1 and consequently a dramatic reduction of nephrin expression. Recruitment of the groucho-related protein TLE4 may be involved in converting Pax2 into a transcriptional repressor of Wt1. Finally, treatment of mice with an angiotensin-converting enzyme (ACE) inhibitor normalized renal function and induced upregulation of the important structural molecule nephrin via a Wt1-independent pathway. Our data demonstrate the functional significance of PAX2 reexpression in mature podocytes for the development of glomerular diseases and suggest that reactivation of PAX genes in terminally differentiated cells leads to a more dedifferentiated phenotype.

Bone marrow transplantation can attenuate the progression of mesangial sclerosis.

Bone marrow (BM) transplantation has been shown to provide beneficial effects in injured organs, including heart, liver, and kidney. We explored the therapeutic potential of BM transplantation (BMT) in Wilms' tumor suppressor 1 (Wt1) heterozygous mice, which represent a model of mesangial sclerosis. After transplantation of wild-type BM, there is statistically significantly lower urinary albumin and increased survival in Wt1+/- recipients. Control BMT using Wt1+/- donors showed no significant beneficial effects. The long-term beneficial effect of BMT was dependent on the dose of irradiation applied to the recipients before BMT. At a lethal dose of 1,000 cGy, the decrease in albuminuria and prolongation of lifespan in Wt1+/- mice were transient, with maximal amelioration at 12 weeks and resumption of albuminuria by 24 weeks after BMT. This was, at least in part, due to irradiation and not Wt1 heterozygosity because wild-type recipients also developed albuminuria within 24 weeks of BMT with 1,000 cGy. In contrast, Wt1+/- mice transplanted after 400 cGy showed long-term improvement in albuminuria and lifespan. Approximately 0.4% of podocytes were marrow derived, a level that is unlikely to be responsible for the therapeutic effects. In addition, donor BM cells formed rings surrounding the glomeruli, and approximately one third of the cells in these rings were macrophages. In conclusion, transplantation of wild-type BM attenuates progression of mesangial sclerosis in the Wt1+/- model of renal disease, and the mechanism by which this occurs may involve engraftment of BM-derived cells in the renal parenchyma.

Early gonadal development: exploring Wt1 and Sox9 function.

Prior to sex determination the gonadal anlage is formed as a bipotential primordium with the capacity to differentiate into either testes or ovaries depending on the presence or absence of the Sry gene. Knockout experiments have implicated five genes in the formation or survival of the gonadal primordium: Wt1, Sf1, Lim1, Lhx9 and Emx2. We are particularly interested in the Wilms tumour suppressor, WT1, which is characterized by complex posttranscriptional modifications. Here we will focus on published in vitro evidence suggesting distinct functions for the various isoforms and present our own results from in vivo experiments. Our data suggest that WT1 is an important regulator of the transcription or stability of the sex-determining gene Sry. One of the first genes expressed after the initial male sex-determining signal is the Sox9 gene. Human SOX9 has been implicated in male-to-female sex reversal. To analyse Sox9 function in mouse development we have performed transgenic experiments and ectopically expressed this gene in XX gonads. Our data indicate that Sox9 is sufficient to induce testis formation in mice. Here we will discuss our new data and present an updated model for Wt1 and Sox9 function in gonad formation and sex determination.

WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis.

Glomerular disease is one of the most common causes of end-stage renal failure. Increasing evidence suggests that these glomerulopathies are frequently caused by primary lesions in the renal podocytes. One of the major consequences of podocyte lesions is the accumulation of mesangial matrix in the glomerular basement membrane, a process called glomerulosclerosis. Mesangial sclerosis is one of the most consistent findings in Denys-Drash patients and can be caused by dominant mutations in the Wilms' tumor 1 gene (WT1). The underlying mechanism, however, is poorly understood. WT1 is expressed in the podocytes throughout life, but its function in this cell type is unknown. Combining Wt1-knockout and inducible yeast artificial chromosome transgenic mouse models, we demonstrate that reduced expression levels of WT1 result in either crescentic glomerulonephritis or mesangial sclerosis depending on the gene dosage. Strikingly, the two podocyte-specific genes nphs1 and podocalyxin are dramatically downregulated in mice with decreased levels of Wt1, suggesting that these two genes act downstream of Wt1. Taken together, our data provide genetic evidence that reduced levels of Wt1 are responsible for the pathogenesis of two distinct renal diseases and offer a molecular explanation for the increased occurrence of glomerulosclerosis in patients with WAGR syndrome.