The Preserving Kidney Function in Children With CKD (PRESERVE) Study: Rationale, Design, and Methods.

Rationale & Objective: PRESERVE seeks to provide new knowledge to inform shared decision-making regarding blood pressure (BP) management for pediatric chronic kidney disease (CKD). PRESERVE will compare the effectiveness of alternative strategies for monitoring and treating hypertension on preserving kidney function; expand the National Patient-Centered Clinical Research Network (PCORnet) common data model by adding pediatric- and kidney-specific variables and linking electronic health record data to other kidney disease databases; and assess the lived experiences of patients related to BP management. Study Design: Multicenter retrospective cohort study (clinical outcomes) and cross-sectional study (patient-reported outcomes [PROs]). Setting & Participants: PRESERVE will include approximately 20,000 children between January 2009-December 2022 with mild-moderate CKD from 15 health care institutions that participate in 6 PCORnet Clinical Research Networks (PEDSnet, STAR, GPC, PaTH, CAPRiCORN, and OneFlorida+). The inclusion criteria were ≥1 nephrologist visit and ≥2 estimated glomerular filtration rate (eGFR) values in the range of 30 to <90 mL/min/1.73 m2 separated by ≥90 days without an intervening value ≥90 mL/min/1.73 m2 and no prior dialysis or kidney transplant. Exposures: BP measurements (clinic-based and 24-hour ambulatory BP); urine protein; and antihypertensive treatment by therapeutic class. Outcomes: The primary outcome is a composite event of a 50% reduction in eGFR, eGFR of <15 mL/min/1.73 m2, long-term dialysis or kidney transplant. Secondary outcomes include change in eGFR, adverse events, and PROs. Analytical Approach: Longitudinal models for dichotomous (proportional hazards or accelerated failure time) and continuous (generalized linear mixed models) clinical outcomes; multivariable linear regression for PROs. We will evaluate heterogeneity of treatment effect by CKD etiology and degree of proteinuria and will examine variation in hypertension management and outcomes based on socio-demographics. Limitations: Causal inference limited by observational analyses. Conclusions: PRESERVE will leverage the PCORnet infrastructure to conduct large-scale observational studies that address BP management knowledge gaps for pediatric CKD, focusing on outcomes that are meaningful to patients. Plain-Language Summary: Hypertension is a major modifiable contributor to loss of kidney function in chronic kidney disease (CKD). The purpose of PRESERVE is to provide evidence to inform shared decision-making regarding blood pressure management for children with CKD. PRESERVE is a consortium of 16 health care institutions in PCORnet, the National Patient-Centered Clinical Research Network, and includes electronic health record data for >19,000 children with CKD. PRESERVE will (1) expand the PCORnet infrastructure for research in pediatric CKD by adding kidney-specific variables and linking electronic health record data to other kidney disease databases; (2) compare the effectiveness of alternative strategies for monitoring and treating hypertension on preserving kidney function; and (3) assess the lived experiences of patients and caregivers related to blood pressure management.

Correction to: Predictors of patency for arteriovenous fistulae and grafts in pediatric hemodialysis patients.

The original version of this article unfortunately contained a mistake. The name of Vimal Chadha was presented incorrectly. The corrected author list is given above.

Predictors of patency for arteriovenous fistulae and grafts in pediatric hemodialysis patients.

BACKGROUND: Hemodialysis (HD) guidelines recommend permanent vascular access (PVA) in children unlikely to receive kidney transplant within 1 year of starting HD. We aimed to determine predictors of primary and secondary patency of PVA in pediatric HD patients. METHODS: Retrospective chart reviews were performed for first PVAs in 20 participating centers. Variables collected included patient demographics, complications, interventions, and final outcome. RESULTS: There were 103 arterio-venous fistulae (AVF) and 14 AV grafts (AVG). AVF demonstrated superior primary (p = 0.0391) and secondary patency (p = 0.0227) compared to AVG. Primary failure occurred in 16 PVA (13.6%) and secondary failure in 14 PVA (12.2%). AVF were more likely to have primary failure (odds ratio (OR) = 2.10) and AVG had more secondary failure (OR = 3.33). No demographic, clinical, or laboratory variable predicted primary failure of PVA. Anatomical location of PVA was predictive of secondary failure, with radial having the lowest risk compared to brachial (OR = 12.425) or femoral PVA (OR = 118.618). Intervention-free survival was predictive of secondary patency for all PVA (p = 0.0252) and directly correlated with overall survival of AVF (p = 0.0197) but not AVG. Study center demonstrated statistically significant effect only on intervention-free AVF survival (p = 0.0082), but not number of complications or interventions, or outcomes. CONCLUSIONS: In this multi-center pediatric HD cohort, AVF demonstrated primary and secondary patency advantages over AVG. Radial PVA was least likely to develop secondary failure. Intervention-free survival was the only predictor of secondary patency for AVF and directly correlated with overall access survival. The study center effect on intervention-free survival of AVF deserves further investigation.

Microarrays and RNA-Seq identify molecular mechanisms driving the end of nephron production.

BACKGROUND: The production of nephrons suddenly ends in mice shortly after birth when the remaining cells of the multi-potent progenitor mesenchyme begin to differentiate into nephrons. We exploited this terminal wave of nephron production using both microarrays and RNA-Seq to serially evaluate gene transcript levels in the progenitors. This strategy allowed us to define the changing gene expression states following induction and the onset of differentiation after birth. RESULTS: Microarray and RNA-Seq studies of the progenitors detected a change in the expression profiles of several classes of genes early after birth. One functional class, a class of genes associated with cellular proliferation, was activated. Analysis of proliferation with a nucleotide analog demonstrated in vivo that entry into the S-phase of the cell cycle preceded increases in transcript levels of genetic markers of differentiation. Microarrays and RNA-Seq also detected the onset of expression of markers of differentiation within the population of progenitors prior to detectable Six2 repression. Validation by in situ hybridization demonstrated that the markers were expressed in a subset of Six2 expressing progenitors. Finally, the studies identified a third set of genes that provide indirect evidence of an altered cellular microenvironment of the multi-potential progenitors after birth. CONCLUSIONS: These results demonstrate that Six2 expression is not sufficient to suppress activation of genes associated with growth and differentiation of nephrons. They also better define the sequence of events after induction and suggest mechanisms contributing to the rapid end of nephron production after birth in mice.

Defining the genetic blueprint of kidney development.

Thousands of genes show differential expression patterns during kidney development, suggesting that the genetic program driving this process is complex. While great progress has been made in defining the outline of the genetic basis of nephrogenesis, it is clear that much remains to be learned. A global atlas of the gene expression profiles of the multiple elements of the developing kidney would allow the identification of novel growth factor-receptor interactions, identify additional molecular markers of distinct components, facilitate the generation of compartment specific GFP-CRE transgenic mouse tools, lend insights into the genetic regulatory circuits governing nephron formation, and fully characterize the waves of gene expression that impel nephrogenesis. Both microarrays and next generation deep sequencing of cDNA libraries can be used to define comprehensive, sensitive, and quantitative gene expression profiles. In addition, laser capture microdissection and transgenic GFP mice can be used to isolate specific compartments and pure cell types from the developing kidney. Advancing technologies are even allowing robust gene expression profiling of single cells. The final goal is the production of an exquisitely detailed atlas of the gene expression program that drives kidney development.

Microdissection of the gene expression codes driving nephrogenesis.

The kidney represents an excellent model system for learning the principles of organogenesis. It is intermediate in complexity, and employs many commonly used developmental processes. As such, kidney development has been the subject of intensive study, using a variety of techniques, including in situ hybridization, organ culture and gene targeting, revealing many critical genes and pathways. Nevertheless, proper organogenesis requires precise patterns of cell type specific differential gene expression, involving very large numbers of genes. This review is focused on the use of global profiling technologies to create an atlas of gene expression codes driving development of different mammalian kidney compartments. Such an atlas allows one to select a gene of interest, and to determine its expression level in each element of the developing kidney, or to select a structure of interest, such as the renal vesicle, and to examine its complete gene expression state. Novel component specific molecular markers are identified, and the changing waves of gene expression that drive nephrogenesis are defined. As the tools continue to improve for the purification of specific cell types and expression profiling of even individual cells it is possible to predict an atlas of gene expression during kidney development that extends to single cell resolution.

Atlas of gene expression in the developing kidney at microanatomic resolution.

Kidney development is based on differential cell-type-specific expression of a vast number of genes. While multiple critical genes and pathways have been elucidated, a genome-wide analysis of gene expression within individual cellular and anatomic structures is lacking. Accomplishing this could provide significant new insights into fundamental developmental mechanisms such as mesenchymal-epithelial transition, inductive signaling, branching morphogenesis, and segmentation. We describe here a comprehensive gene expression atlas of the developing mouse kidney based on the isolation of each major compartment by either laser capture microdissection or fluorescence-activated cell sorting, followed by microarray profiling. The resulting data agree with known expression patterns and additional in situ hybridizations. This kidney atlas allows a comprehensive analysis of the progression of gene expression states during nephrogenesis, as well as discovery of potential growth factor-receptor interactions. In addition, the results provide deeper insight into the genetic regulatory mechanisms of kidney development.

Cessation of renal morphogenesis in mice.

The kidney develops by cycles of ureteric bud branching and nephron formation. The cycles begin and are sustained by reciprocal inductive interactions and feedback between ureteric bud tips and the surrounding mesenchyme. Understanding how the cycles end is important because it controls nephron number. During the period when nephrogenesis ends in mice, we examined the morphology, gene expression, and function of the domains that control branching and nephrogenesis. We found that the nephrogenic mesenchyme, which is required for continued branching, was gone by the third postnatal day. This was associated with an accelerated rate of new nephron formation in the absence of apoptosis. At the same time, the tips of the ureteric bud branches lost the typical appearance of an ampulla and lost Wnt11 expression, consistent with the absence of the capping mesenchyme. Surprisingly, expression of Wnt9b, a gene necessary for mesenchyme induction, continued. We then tested the postnatal day three bud branch tip and showed that it maintained its ability both to promote survival of metanephric mesenchyme and to induce nephrogenesis in culture. These results suggest that the sequence of events leading to disruption of the cycle of branching morphogenesis and nephrogenesis began with the loss of mesenchyme that resulted from its conversion into nephrons.

Laser capture-microarray analysis of Lim1 mutant kidney development.

The Lim1 gene has essential functions during several stages of kidney development. In particular, a tissue-specific knockout in the early metanephric mesenchyme results in the formation of the earliest nephron precursor, the renal vesicle, but failure of this structure to progress to the next stage, the comma-shaped body. To better understand the molecular nature of this developmental arrest, we used a laser capture microdissection-microarray strategy to examine the perturbed gene expression pattern of the mutant renal vesicles. Among the genes found differently expressed were Chrdl2, an inhibitor of BMP signaling, the proapoptotic factor Bmf, as well as myob5, an atypical myosin that modulates chemokine signaling, and pdgfrl, which is important in epithelial folding. Of particular interest, the microarray data indicated that the Dkk1 gene, which encodes an inhibitor of Wnt signaling, was downregulated ninefold in mutants. This was confirmed by in situ hybridizations. It is interesting to note that Lim1 and Dkk1 mutant mice have striking similarities in phenoytpe. These results suggest that the Dkk1 gene might be a key downstream effector of Lim1 function.

Pygo1 and Pygo2 roles in Wnt signaling in mammalian kidney development.

BACKGROUND: The pygopus gene of Drosophila encodes an essential component of the Armadillo (beta-catenin) transcription factor complex of canonical Wnt signaling. To better understand the functions of Pygopus-mediated canonical Wnt signaling in kidney development, targeted mutations were made in the two mammalian orthologs, Pygo1 and Pygo2. RESULTS: Each mutation deleted >80% of the coding sequence, including the critical PHD domain, and almost certainly resulted in null function. Pygo2 homozygous mutants, with rare exception, died shortly after birth, with a phenotype including lens agenesis, growth retardation, altered kidney development, and in some cases exencephaly and cleft palate. Pygo1 homozygous mutants, however, were viable and fertile, with no detectable developmental defects. Double Pygo1/Pygo2 homozygous mutants showed no apparent synergy in phenotype severity. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced levels of expression in Pygo1-/-/Pygo2-/- mutants, with tissue-specific variation in degree of diminution. The Pygo1 and Pygo2 genes both showed widespread expression in the developing kidney, with raised levels in the stromal cell compartment. Confocal analysis of the double mutant kidneys showed disturbance of both the ureteric bud and metanephric mesenchyme-derived compartments. Branching morphogenesis of the ureteric bud was altered, with expanded tips and reduced tip density, probably contributing to the smaller size of the mutant kidney. In addition, there was an expansion of the zone of condensed mesenchyme capping the ureteric bud. Nephron formation, however, proceeded normally. Microarray analysis showed changed expression of several genes, including Cxcl13, Slc5a2, Klk5, Ren2 and Timeless, which represent candidate Wnt targets in kidney development. CONCLUSION: The mammalian Pygopus genes are required for normal branching morphogenesis of the ureteric bud during kidney development. Nevertheless, the relatively mild phenotype observed in the kidney, as well as other organ systems, indicates a striking evolutionary divergence of Pygopus function between mammals and Drosophila. In mammals, the Pygo1/Pygo2 genes are not absolutely required for canonical Wnt signaling in most developing systems, but rather function as quantitative transducers, or modulators, of Wnt signal intensity.

pygopus 2 has a crucial, Wnt pathway-independent function in lens induction.

Drosophila Pygopus was originally identified as a core component of the canonical Wnt signaling pathway and a transcriptional coactivator. Here we have investigated the microophthalmia that arises in mice with a germline null mutation of pygopus 2. We show that this phenotype is a consequence of defective lens development at inductive stages. Using a series of regionally limited Cre recombinase transgenes for conditional deletion of Pygo2(flox), we show that Pygo2 activity in pre-placodal presumptive lens ectoderm, placodal ectoderm and ocular mesenchyme all contribute to lens development. In each case, Pygo2 is required for normal expression levels of the crucial transcription factor Pax6. Finally, we provide multiple lines of evidence that although Pygo2 can function in the Wnt pathway, its activity in lens development is Wnt pathway-independent.

Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development.

The Hox11 paralogous genes play critical roles in kidney development. They are expressed in the early metanephric mesenchyme and are required for the induction of ureteric bud formation and its subsequent branching morphogenesis. They are also required for the normal nephrogenesis response of the metanephric mesenchyme to inductive signals from the ureteric bud. In this report, we use microarrays to perform a comprehensive gene expression analysis of the Hoxa11/Hoxd11 mutant kidney phenotype. We examined E11.5, E12.5, E13.5 and E16.5 developmental time points. A novel high throughput strategy for validation of microarray data is described, using additional biological replicates and an independent microarray platform. The results identified 13 genes with greater than 3-fold change in expression in early mutant kidneys, including Hoxa11s, GATA6, TGFbeta2, chemokine ligand 12, angiotensin receptor like 1, cytochrome P450, cadherin5, and Lymphocyte antigen 6 complex, Iroquois 3, EST A930038C07Rik, Meox2, Prkcn, and Slc40a1. Of interest, many of these genes, and others showing lower fold expression changes, have been connected to processes that make sense in terms of the mutant phenotype, including TGFbeta signaling, iron transport, protein kinase C function, growth arrest and GDNF regulation. These results identify the multiple molecular pathways downstream of Hox11 function in the developing kidney.

Induction of renal tubular cell apoptosis in focal segmental glomerulosclerosis: roles of proteinuria and Fas-dependent pathways.

The hypothesis that apoptosis represents a proximate mechanism by which tubule cells are damaged in FSGS was tested. Thirty kidney biopsy specimens from children with idiopathic early FSGS were studied retrospectively. Unexpected, apoptosis was evident in both proximal and distal tubule cells. There was a significant correlation between the degree of proteinuria and the number of apoptotic cells. Fas protein was detected predominantly in the tubule cells that underwent apoptosis. When compared with patients with other chronic proteinuric states, those with FSGS displayed a proliferation/apoptosis ratio in favor of proliferation in the glomerulus but dramatically in favor of apoptosis in the tubules. When both proteinuria and apoptosis were included in a stepwise logistic regression procedure, only apoptosis was found to predict independently the development of ESRD. Prolonged incubation of cultured Madin-Darby canine kidney (distal/collecting) cells with albumin also resulted in a dose- and duration-dependent induction of apoptosis and activation of the Fas pathway, lending support to the novel finding of distal tubule cell apoptosis in patients with FSGS. The results indicate that an elevated tubule cell apoptosis rate at the time of initial biopsy represents an independent predictor of progression to ESRD in patients with early FSGS.

Microarray analysis of focal segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a leading cause of chronic renal failure in children. Recent studies have begun to define the molecular pathogenesis of this heterogeneous condition. Here we use oligonucleotide microarrays to obtain a global gene expression profile of kidney biopsy specimens from patients with FSGS in order to better understand the pathogenesis of this disease. METHODS: We extracted RNA from renal biopsy samples of 10 patients with the diagnosis of FSGS and from 5 control kidney samples, and produced labeled cRNA for hybridization to Affymetrix human U133A microarrays. RESULTS: We identified a gene expression fingerprint for FSGS that contained 429 of 22,283 possible genes, each with a p < 0.01, using RMA normalization, Welch t test, and at least a 1.8-fold change in 5 of the 10 patients examined. We also found gene expression differences in samples from subsets of patients who had either nephrotic syndrome or renal insufficiency. This screen identified many genes and genetic pathways that have already been implicated in the pathogenesis of FSGS. In addition, we found changes in gene expression in genetic pathways that have not been studied in FSGS. CONCLUSIONS: Oligonucleotide DNA microarray analysis of renal biopsy specimens identified a gene expression fingerprint in samples from a heterogeneous population of patients with FSGS. The genes and genetic pathways identified in this study can be compared to results of similar studies of other diseases to examine specificity and used to study the pathogenesis of FSGS.

Atlas of Hox gene expression in the developing kidney.

Hox genes often play important roles in segment identity determination and organogenesis. To better understand the roles of Hox genes during kidney development, we performed an extensive analysis of their expression patterns. Section in situ hybridizations were used to define the expression of 37 Hox genes at embryonic day (E) 12.5, E13.5, E15.5, and E17.5 of kidney development. Several interesting principles emerged. First, the concept of colinearity was preserved. Hox genes from the more 3' positions in clusters were more often expressed in the ureteric bud, which is derived from the anterior of the intermediate mesoderm. Second, Hox genes were expressed throughout the ureteric bud without any segment specificity. Third, in the different segments of the forming nephron we did observe overlapping domains of Hox gene expression, which initiated distally at the junction between the nephron and ureteric bud, and extended proximally variable distances. Finally, we observed that paralogous Hox genes often showed surprisingly diverse expression patterns. Indeed, contiguous genes on a single cluster more often showed similar expression patterns than paralogs. In summary, the resulting atlas of Hox gene expression provides a foundation for further study of the overlapping functions Hox genes in the developing kidney.

Profiling gene expression in kidney development.

Rapid determination of the expression levels of all genes can be achieved with microarrays. This technology is beginning to revolutionize the study of kidney development. Genome-wide expression studies of the developing rat, mouse and human kidneys have found thousands of expressed genes, with many hundreds showing changes in expression as a function of developmental time. The resulting gene expression profiles provide an important discovery function, identifying new genes and pathways not previously implicated in kidney organogenesis. In combination with microdissection techniques, microarrays further extend global gene expression analysis to discrete components or cell types within the kidney. The resulting detailed molecular portrait of normal kidney development provides a baseline for studies of the many mouse mutants available with abnormal kidney development.

Gene expression in early ischemic renal injury: clues towards pathogenesis, biomarker discovery, and novel therapeutics.

Acute renal failure (ARF) represents a common and serious problem in clinical medicine. Renal ischemia-reperfusion injury (IRI) is the major cause of ARF in the native and transplanted kidney. Several decades of research have provided successful therapeutic approaches in animal models, but translational efforts in humans have yielded disappointing results. The major reasons for this include a lack of early markers for ARF (and hence a delay in initiating therapy), and the multi-factorial nature of the disease. This review focuses on the use of cDNA microarrays to elucidate the molecular genetic mechanisms underlying tubule cell apoptosis, and to identify novel biomarkers for early renal IRI. Also presented is a comparative temporal analysis of cDNA microarray results from mature kidneys following IRI and during normal nephrogenesis. Molecular genetic evidence for the notion that regeneration recapitulates development in the kidney, and that injured tubule cells possess the capacity to de-differentiate to the earliest stages of development, is presented. The implications of these findings to the ability of the kidney to repair itself and potential strategies for accelerating recovery are briefly discussed.

A catalogue of gene expression in the developing kidney.

BACKGROUND: Although many genes with important function in kidney morphogenesis have been described, it is clear that many more remain to be discovered. Microarrays allow a more global analysis of the genetic basis of kidney organogenesis. METHODS: In this study, Affymetrix U74Av2 microarrays, with over 12,000 genes represented, were used in conjunction with robust target microamplification techniques to define the gene expression profiles of the developing mouse kidney. RESULTS: Microdissected murine ureteric bud and metanephric mesenchyme as well as total kidneys at embryonic day E11.5, E12.5, E13.5, E16.5, and adult were examined. This work identified, for example, 3847 genes expressed in the E12.5 kidney. Stringent comparison of the E12.5 versus adult recognized 428 genes with significantly elevated expression in the embryonic kidney. These genes fell into several functional categories, including transcription factor, growth factor, signal transduction, cell cycle, and others. In contrast, surprisingly few differences were found in the gene expression profiles of the ureteric bud and metanephric mesenchyme, with many of the differences clearly associated with the more epithelial character of the bud. In situ hybridizations were used to confirm and extend microarray-predicted expression patterns in the developing kidney. For three genes, Cdrap, Tgfbi, and Col15a1, we observed strikingly similar expression in the developing kidneys and lungs, which both undergo branching morphogenesis. CONCLUSION: The results provide a gene discovery function, identifying large numbers of genes not previously associated with kidney development. This study extends developing kidney microarray analysis to the powerful genetic system of the mouse and establishes a baseline for future examination of the many available mutants. This work creates a catalogue of the gene expression states of the developing mouse kidney and its microdissected subcomponents.

Hox genes and kidney patterning.

PURPOSE OF REVIEW: Hox gene activity is essential for proper organization or pattern of the vertebrate body plan and is necessary for organogenesis. Sequence conservation within this family of genes is high yet they are involved in very diverse developmental processes. How this family functions in these processes is a challenging question, but is important for the understanding of renal organogenesis. Multiple Hox genes are expressed in the kidney and mutation in at least one group of paralogous genes results in severe renal defects. RECENT FINDINGS: Recent studies in mice with targeted Hox gene mutations and in kidney cell lines demonstrate that these genes have evolved to control tissue specific functions through their ability to regulate the expression of renal morphogens. The studies also demonstrate that Hox gene activity is not only restricted by the domain of expression but also by the specificity of the DNA binding homeodomain. Interestingly, these conserved homeodomains are not wholly interchangeable for normal renal organogenesis while they do appear to be interchangeable for axial skeleton development. SUMMARY: It is clear that Hox genes regulate important interactions between the ureteric bud and metanephric mesenchyme. Nevertheless, much work remains to define the expression patterns of multiple Hox genes during kidney development, to better determine the functional relationships of the encoded proteins, and to identify additional Hox downstream targets.

Hoxa 11 is upstream of Integrin alpha8 expression in the developing kidney.

Mutation of the functionally redundant Hoxa 11/Hoxd 11 genes gives absent or rudimentary kidneys resulting from a dramatic reduction of the growth and branching of the ureteric bud. To understand better the molecular mechanisms of Hoxa 11/Hoxd 11 function in kidney development, it is necessary to identify the downstream target genes regulated by their encoded transcription factors. To this end, we conducted a screen for Hoxa 11-responsive genes in two kidney cell lines. HEK293 cells, which usually do not express Hoxa 11, were modified to allow inducible Hoxa 11 expression. The mK10 cells, derived specifically for this study from Hoxa 11/Hoxd 11 double-mutant mice, were also modified to give cell populations with and without Hoxa 11 expression. Differential display, Gene Discovery Arrays, and Affymetrix genechip probe arrays were used to screen for genes up- or down-regulated by Hoxa 11. Nine genes, PDGF A, Cathepsin L, annexin A1, Mm.112139, Est2 repressor factor, NrCAM, ZNF192, integrin-associated protein, and GCM1, showed reproducible 3-fold or smaller changes in gene expression in response to Hoxa 11. One gene, the Integrin alpha8, was up-regulated approximately 20-fold after Hoxa 11 expression. The Integrin alpha8 gene is expressed together with Hoxa 11 in metanephric mesenchyme cells, and mutation of Integrin alpha8 gives a bud-branching morphogenesis defect very similar to that observed in Hoxa 11/Hoxd 11 mutant mice. In situ hybridizations showed a dramatic regional reduction in Integrin alpha8 expression in the developing kidneys of Hoxa 11/Hoxd 11 mutant mice. This work suggests that the Integrin alpha8 gene may be a major effector of Hoxa 11/Hoxd 11 function in the developing kidney.