Hepatocyte Nuclear Factor 1 and Hypertensive Nephropathy

Animals

Studies were performed on 4-, 8-, 12-, and 18-week-old male animals (n=3 to 4 per strain per age). We used WKY and injury-prone spontaneously hypertensive-A3 (SHR-A3) rats (derived from stocks previously maintained in Heidelberg and kindly provided by Dr Klaus Lindpaintner, Brigham and Women’s Hospital, Boston, Mass) that have been maintained in our facility for 10 years. We also used injury-resistant SHR-B2 and SHR-C animals bred from stocks kindly provided by Professor T. Suzuki, Kinki University School of Medicine, Japan. The genealogical relationships among SHR lines and between SHR and WKY have been documented, and these 3 lines represent the 3 main breeding lines used to generate inbred SHR animals in Japan.^2,3,14 More comprehensive information on the relations among SHR lines can be obtained at the Rat Genome Database (http://rgd.mcw.edu/strains) and the National Bio Resource Project for the Rat in Japan (http://www.anim.med.kyoto-u.ac.jp/nbr/home.htm). Animals were housed under controlled conditions in an AAALAC-approved animal facility and provided a standard rodent chow diet and drinking water ad libitum. No dietary sodium loading was used. All animal use was prospectively reviewed and approved by the University’s Animal Welfare Committee.

Blood pressure in male SHR-A3 (n=16) and male SHR-B2 (n=16) was measured by radio-telemetry (Data Sciences) in adult animals 16 to 19 weeks of age. Telemetry catheters were implanted in the abdominal aorta in animals under isoflurane anesthesia. Recordings were begun at least 7 days after implantation and continued for 5 to 7 days. Differences in blood pressure between SHR-A3 and SHR-B2 were sought by single factor analysis of variance.

For tissue collection, animals were anesthetized by isoflurane inhalation and kidneys were rapidly dissected via ventral laparotomy. Renal gene expression analysis used total RNA preparations from axial renal segments including cortex and medulla. Ureteric pelvis and major vascular structures of the renal sinus were removed from the sample. Each sample from each animal was treated as an independent sample, and no pooling was performed.

Histological Analysis

Kidney tissue was fixed in 4% buffered formalin and paraffin embedded using standard techniques. Five-micron serial sections were stained with Periodic-Acid-Schiff stain (PAS) for assessment of renal injury and PicroSirus red to evaluate the extent of collagen accumulation.¹⁵ Histological analysis was performed in a blinded fashion.

Gene Array Analysis

Initial studies were focused on expression of genes involved in pathways of reactive radical formation and detoxification. Affymetrix rat RG-U34A arrays were used to determine transcript abundance of genes in SHR-A3, SHR-B2, SHR-C, and WKY animals in each of the 4 age groups. Three to 4 replicate samples per line×age group were included in the experiments. Sample preparation, array hybridization, and array scanning were performed according to the manufacturer’s recommended protocols. Data processing and scaling before analysis was performed using the Affymetrix MAS5.0 software. This data set has been deposited in the NCBI Gene Expression Omnibus (GEO database) with series accession number GSE2104 and was previously used to seek evidence of the heritability of gene expression in SHR lines.¹⁶

TaqMan Assays to Verify Array Data

We performed quantitative RT-PCR to verify gene expression differences observed by gene array. Comparisons were made in renal RNA collected from SHR-A3 and WKY animals. The analysis used an ABI TaqMan 7700 instrument with reporter probes coupled at the 5′ terminus to FAM and at the 3′ terminus to TAMRA. Normalization of transcript abundance in each sample was by comparison with abundance of 18S RNA.

Electrophoretic Mobility Shift Assays

Gel shift assays were performed using double stranded (DS) oligos corresponding to proximal promoter regions containing putative HNF1 matrix sequences. Nuclear extracts were prepared from rat kidney cells. DNA DS oligos were labeled with ³²P. After incubation with nuclear extracts, reactions were resolved on acrylamide gels and developed on film. Competitions were performed with excess unlabeled DS oligos, these included DS oligos with the same sequence as the labeled oligo, DS oligos that were otherwise identical except for containing mutations in residues conserved in the HNF1 binding matrix and DS oligos containing mutations outside the HNF1 matrix.

Bioinformatics

After identification of a set of genes involved in redox balance (please see Table S1, available online at http://hyper.ahajournals.org), features of the proximal control of expression of these genes were analyzed in silico using the Genomatix software, MatInspector, to identify the occurrence and frequency of transcription factor matrix sequences in the proximal promoters of redox balance genes (www.genomatix.de).

Emergence of Hypertensive Renal Injury in SHR-A3 Occurs in the Absence of Dietary Modification

Although SHR-A3 is recognized to be prone to end-organ injury, this susceptibility has been reported to require a “Japanese-style” diet with elevated sodium and reduced potassium and protein intake.¹⁷ Using PAS and Picro-Sirius red staining we observed that renal structures were undamaged and fibrosis was absent in 18 weeks old SHR-A3 raised on a standard laboratory rodent chow and provided water to drink (Figure 1). However, by 24 weeks of age, glomerular and tubular injury, along with fibrosis and increased interstitial cellularity were present in SHR-A3, but absent from WKY and from the injury resistant SHR-B2 and SHR-C strains. The extent and severity of this injury progressed in SHR-A3 animals at 30 weeks of age, but the other SHR lines remained free from injury.

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Figure 1. A, Periodic Acid-Schiff stained sections of kidney from 4 rat lines (WKY, SHR-A3, SHR-B2, SHR-C, columns) at 18, 24, and 30 weeks of age (rows). SHR-A3 demonstrates patchy injury at 24 weeks of age that is absent at 18 weeks of age and that increases in extent and severity by 30 weeks of age. Injury includes glomerular hypercellularity and sclerosis, as well as tubulointerstial inflammation and fibrosis. No similar evidence of renal injury was present in other rats lines. Magnification is eyepiece 10×, objective 20×. B, Picric-acid-Sirius red staining of similar sections as in A. Sirius red stains collagen and provides an indication of fibrotic change. Evidence of increased glomerular and tubular collagen accumulation emerges in the 24-week-old SHR-A3 and is largely absent from the other lines which remain free of fibrotic change at least up to 30 weeks of age.

Studies by others produced no clear consensus whether blood pressure differs between SHR-A3 and injury-resistant SHR lines.^4,18 However, this issue has not been rigorously examined using precise radio-telemetry methods for measuring blood pressure. We made such measurements in SHR-A3 and SHR-B2 animals, recording blood pressure after the maturation of hypertension, but before the emergence of renal injury (16 to 19 weeks of age). We observed a small, but statistically significant difference in blood pressure (please see Table S2).

Expression of Redox Genes Is Altered in SHR-A3 Before the Emergence of Renal Injury

Renal redox stress may contribute to renal injury in hypertension. This suggested the utility of examining changes in gene expression along pathways involved in radical production and mitigation during the development of hypertension in SHR lines that contrast in susceptibility to renal injury. We performed a literature survey and identified 67 genes whose protein products have been implicated either in the processes by which oxidative species are generated or detoxified (please see Table S1). Representation of these genes on the RG U34A arrays used in the present studies was broad but incomplete (36 of 67 genes). We studied gene expression before the onset of histologically verifiable renal injury, so as to capture changes that may contribute to the initiation of renal injury while avoiding alterations in expression occurring in response to injury.

To assess whether expression of genes involved in redox balance is broadly altered in the renal injury-prone SHR-A3 line, we examined the correlation between expression of individual redox genes in SHR-A3 and the expression of these genes in the other groups of rats (please see Figure S1 for illustration). Table 1 indicates the result of this analysis comparing SHR-A3 to all other strains at each of the 4 ages examined. The strong correlation in expression levels among these redox balance genes across rat lines was remarkably eroded in 18-week-old injury-prone SHR-A3 animals, just before the emergence of histological renal injury. This suggested a major shift in the regulation of redox balance in 18-week-old SHR-A3 compared with injury-resistant strains. We examined the expression of individual redox genes to assess whether any simple patterns among the genes (radical generating genes predominantly up or down, or detoxification genes up or down) might account for the reduced relationship between SHR-A3 redox pathway gene expression. Many genes in pathways involved in radical detoxification were expressed at substantially lower levels in 18-week SHR-A3, but there was no evidence for changes in expression of genes involved in radical generation at 18 weeks of age (though such genes are not as well represented on the array). Among genes involved in radical detoxification we segregated those genes that were downregulated in 18-week-old SHR-A3, compared with WKY, SHR-B2, and SHR-C. We found that 16 of 28 genes involved in radical mitigation were downregulated, comparing SHR-A3 with the other strains (P<0.01, ANOVA followed by Tukey HSD test). Overall, this downregulation was to less than 40% of the level in the reference strain (Figure 2). To validate the altered expression in 18-week-old animals, we selected 9 genes to represent the major component pathways of antioxidant defense and reassessed expression by TaqMan-based quantitative RT-PCR (please see Table S3 for details). The results supported the conclusion from the array data of downregulation in SHR-A3 of each one of the genes tested.

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Figure 2. Expression of 16 or 28 genes identified as involved in radical detoxification. For each gene, the expression level for WKY was normalized to 100 and the corresponding value calculated for the remaining rat lines. The group mean value (±SEM) was then calculated across all 16 genes and plotted. ANOVA followed by Tukey HSD test was used to determine differences between SHR-A3, SHR-B2, and SHR-C. Significant differences observed were between SHR-A3 and SHR-B2 and SHR-A3 and SHR-C (P<0.01).

The Transcription Factor Hepatocyte Nuclear Factor 1 Is an Important Regulator of Redox Gene Expression in the Kidney

The reduced expression in many, but not all, genes involved in radical mitigation suggested a coordinated transcriptional mechanism operating in SHR-A3 and shared by the downregulated genes. We sought to identify this transcriptional regulation by examining the proximal promoters of each of the genes involved in radical detoxification to compare whether transcription factor-DNA interaction matrix sequences (TFM) occurred with differing frequency when the 16 downregulated antioxidant genes were compared with the 12 antioxidant genes that were not downregulated. Promoters were examined by MatInspector, and the TFMs identified were tabulated. Table 2 shows TFMs that were disproportionately represented in the downregulated genes. TFMs for the important renal transcription factor hepatocyte nuclear factor 1 (HNF1) were identified in nearly twice as many downregulated radical antioxidant genes than in such genes that were not downregulated. Further, the total occurrence of such TFMs was nearly 5-fold greater in the downregulated antioxidant genes. Generally, the presence of multiple binding sites for a single transcription factor in the promoter of a gene increases the likelihood that the expression of this gene is regulated by the transcription factor.^19,20

The role of HNF1 in the renal regulation of antioxidant genes was unexpected as only one of the genes has previously been reported to be subject to HNF1 regulation.^21,22 The identification of a TFM in a promoter suggests a regulatory role that requires confirmation. Evidence that HNF1 TFMs correspond to evolutionarily conserved regions of the promoter provides further support of a functional role. We used the Consite tool (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite) to assess concordance between location of HNF1 TFMs and evolutionary conservation of the promoter, comparing rat to human promoter sequences.²³ The results of this analysis support the idea that HNF1 provides regulation of expression of these genes. Figure 3 illustrates the output of one such analysis.

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Figure 3. Evolutionary conservation of HNF1 TFMs demonstrated by comparing the human and rat promoters of downregulated antioxidant genes. Catalase is shown for illustration. The location of HNF1 TFMs in the rat promoter is shown (upper horizontal line), conserved blocks in the human catalase promoter are indicated by the broken lower horizontal line. Overall conservation is shown by the graphed line. The rat HNF1 sites correspond to regions of highest sequence conservation between the rat and the human promoter.

To extend the bioinformatic analysis, we performed gel shift experiments on 5 of the downregulated genes to determine whether HNF1 TFM-containing regions of the promoters of these genes could bind renal nuclear extract proteins. In each of the genes tested, evidence was obtained to support the capacity of HNF1 sites in the promoters of these genes to bind renal nuclear proteins and this appeared to depend specifically on sequences within the HNF1 TFM, rather than adjacent sequences (please see Figure S2).

The Effect of Altered HNF1 Control of Gene Expression in SHR-A3 Extends to Nonredox Genes That Are Controlled by HNF1

A large set of hepatic genes has been identified and experimentally confirmed as being subject to HNF1 regulation.²⁴ We determined which of these genes were both present on the array and expressed at a level greater than background in the rat kidney. This identified 35 further HNF1-regulated genes, none of which is involved in redox balance. We examined the expression of these 35 genes in 18-week-old SHR-A3 compared with the other strains. If the inference that HNF1 regulation of expression of radical scavenging genes is altered in 18-week-old SHR-A3 is correct, it must also be generalizable to other renal genes regulated by HNF1. We found that expression of these genes in SHR-A3 was significantly lower than in the other lines (68.0% of WKY level, P<0.05, Tukey HSD, Figure 4). The occurrence and frequency of HNF1 TFMs in the promoters of downregulated redox genes was similar to that in genes known to be regulated by HNF1 (please see Table S4).

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Figure 4. Effects of altered HNF1 regulation of transcription in SHR-A3 kidney are generalized to other genes lying outside pathways of redox balance. We identified 35 genes for which existing evidence of HNF1 regulation existed that were also present on the array and that had renal expression levels higher than background. Normalizing expression of each gene to the corresponding level in WKY, these genes were found to be, on average, expressed at significantly lower levels in SHR-A3 than in SHr-B2 and SHR-C (P<0.05, Tukey HSD test).

HNF1 TFMs are able to bind 2 HNF1 proteins encoded by Tcf1 and Tcf2. In kidney Tcf2 is the more abundant isoform and is exclusively nuclear.^25,26 Dimerization of Tcf1 and Tcf2 is facilitated by another protein, Dcoh.^27–29 Probes for Tcf1, Tcf2, and Dcoh were present on the array, and expression of all 3 genes was significantly reduced in 18-week-old SHR-A3 compared with the other strains (Figure 5).

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Figure 5. Expression of Tcf1, Tcf2, and Dcoh, each encoding proteins that contribute to the HNF1 transcription factor. Expression levels are normalized to WKY expression as above. Reduced expression of HNF1 component genes suggests that altered expression of HNF1-dependent genes may arise, in part from altered availability of the transcription factor protein.

Perspectives

Redox stress has been implicated in the genesis of hypertensive and diabetic renal disease. This study indicates that the SHR-A3 line, an inbred model of hypertension in the rat, experiences hypertensive renal injury without dietary sodium loading. In contrast, other hypertensive SHR lines resist renal injury, in spite of similar levels of hypertension. In SHR-A3, emergence of histological renal injury is preceded by a change in the expression of many renal redox genes, notably those genes involved in antioxidant mechanisms. We have shown that this change is mediated by upstream transcriptional control produced by the abundant renal transcription factor HNF1. Previously, no role of HNF1 in renal redox balance was known. Altered control of expression by HNF1 is further linked to changes in the expression of many genes in pathways outside redox balance. At present it is unclear whether these changes are the immediate cause of renal injury, however a mouse kidney specific knockout of the gene encoding the major renal HNF1 protein, Tcf2, is associated with early renal failure. Thus, these studies propose a novel unifying mechanism linking the initiation of hypertensive renal injury in SHR-A3 to altered renal function mediated by the broad effects of HNF1 on renal gene expression including the expression of antioxidant genes.